U.S. patent number 5,214,191 [Application Number 07/528,967] was granted by the patent office on 1993-05-25 for oxidant sensitive and insensitive aromatic esters as inhibitors of human neutrophil elastase.
This patent grant is currently assigned to Cortech, Inc.. Invention is credited to John C. Cheronis, Gary P. Kirschenheuter, Lyle W. Spruce.
United States Patent |
5,214,191 |
Kirschenheuter , et
al. |
May 25, 1993 |
Oxidant sensitive and insensitive aromatic esters as inhibitors of
human neutrophil elastase
Abstract
2-Phenylalkanoate esters which are useful as inhibitors of human
leukocyte elastase.
Inventors: |
Kirschenheuter; Gary P.
(Arvada, CO), Spruce; Lyle W. (Arvada, CO), Cheronis;
John C. (Lakewood, CO) |
Assignee: |
Cortech, Inc. (Denver,
CO)
|
Family
ID: |
24107964 |
Appl.
No.: |
07/528,967 |
Filed: |
May 22, 1990 |
Current U.S.
Class: |
514/231.8;
514/456; 548/343.5; 548/475; 548/545; 544/171; 549/350; 548/313.7;
560/11; 560/59; 560/56; 560/45; 560/19; 514/452; 514/450; 514/425;
514/399; 514/315; 560/9; 514/532; 560/106; 560/100; 549/362;
544/392; 560/102; 560/105 |
Current CPC
Class: |
C07C
317/22 (20130101); A61P 9/10 (20180101); C07C
45/46 (20130101); A61P 9/08 (20180101); C07D
249/08 (20130101); A61P 43/00 (20180101); C07C
205/43 (20130101); C07C 323/52 (20130101); C07C
323/20 (20130101); A61P 1/02 (20180101); C07C
69/017 (20130101); C07C 317/44 (20130101); C07D
233/56 (20130101); C07C 69/612 (20130101); C07D
295/155 (20130101); C07C 205/56 (20130101); C07C
309/42 (20130101); A61P 11/00 (20180101); C07D
231/12 (20130101); C07C 45/46 (20130101); C07C
49/792 (20130101); C07C 2601/08 (20170501); C07C
2601/04 (20170501); C07C 2601/14 (20170501); C07C
2601/02 (20170501) |
Current International
Class: |
C07C
205/00 (20060101); C07C 205/56 (20060101); C07C
205/43 (20060101); C07C 317/00 (20060101); C07C
309/42 (20060101); C07C 317/22 (20060101); C07C
317/44 (20060101); C07C 309/00 (20060101); C07C
323/00 (20060101); C07C 323/20 (20060101); C07C
323/52 (20060101); C07D 295/155 (20060101); C07C
45/00 (20060101); C07C 45/46 (20060101); C07D
521/00 (20060101); C07C 69/00 (20060101); C07C
69/017 (20060101); C07C 69/612 (20060101); C07D
295/00 (20060101); C07C 069/76 () |
Field of
Search: |
;560/105,9,11,19,45,56,59,100,102,106 ;548/341,475,545 ;544/171,392
;549/350,362 ;514/240,315,399,425,450,452,456,532 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
3954767 |
May 1976 |
Esteve-Subirana |
4115648 |
September 1978 |
Esteve-Subirana |
4567288 |
January 1986 |
Cousse et al. |
4698344 |
October 1987 |
Sasse et al. |
|
Foreign Patent Documents
|
|
|
|
|
|
|
0068998 |
|
Jan 1983 |
|
EP |
|
0183159 |
|
Jun 1986 |
|
EP |
|
2459614 |
|
Nov 1975 |
|
DE |
|
Other References
CA 109 (7):54438m 1988. .
CA 114 (10):88647x 1990. .
CA 105 (17):153057k 1986..
|
Primary Examiner: Killos; Paul J.
Attorney, Agent or Firm: Cushman, Darby & Cushman
Claims
We claim:
1. A compound of the formula ##STR286## wherein: R.sub.1 and
R.sub.2, which may be the same or different, are selected from the
group consisting of hydrogen, alkyl of 1-6 carbons, cycloalkyl of
3-6 carbons or together represent a methylene group
--(CH.sub.2).sub.n -- where n is a whole number of from 1 to 6;
R.sub.3 represents one or more substituents up to five selected
from the group consisting of:
hydrogen, halogen, haloalkyl of 1-12 carbons, alkyl of 1-12
carbons, alkoxy of 1-12 carbons, alkenyl of 2-12 carbons,
cycloalkyl of 3-12 carbons, mono- or dicyclic aryl,
--ZR.sub.5 where Z is O, S, S(O) or SO.sub.2 and R.sub.5 is
hydrogen, alkyl of 1-18 carbons, cycloalkyl of 3-12 carbons or
phenyl;
--NR.sub.6 R.sub.7 where R.sub.6 and R.sub.7 may be the same or
different and may be hydrogen, alkyl of 1-12 carbons, cycloalkyl or
3-6 carbons, phenyl, alkoxy of 1-12 carbons, acyl of the formula
--C(O)R.sub.6 is alkyl of 1-12 carbons, cycloalkyl of 3-12 carbons,
phenyl, CH.sub.3 OC(O)CH.sub.2 CH.sub.2 --, HOOCCH.sub.2 CH.sub.2
--,
NaO.sub.3 SCH.sub.2 CH.sub.2 NHC(O)CH.sub.2 CH.sub.2 --, or R.sub.6
and R.sub.7 together may represent --C(O)CH.sub.2 CH.sub.2 C(O)--,
--C(O)--C.sub.6 H.sub.4 -- C(O)-- or --(CH.sub.2).sub.x -- where x
is 2, 3, 4, 5 and 6;
morpholino, imidazole or piperazino joined to the phenyl ring
through a nitrogen atom; or
R.sub.3 represents alkylene group containing 3 to 4 carbons, or
such group substituted with lower alkoxy or the group
--O(CH.sub.2).sub.n O-- where n is 2, 3 or 4, joining adjacent
carbons of the phenyl ring;
R.sub.4 is from one to five substituents selected from hydrogen,
halogen, nitro, --C(O)CH.sub.3, S(O).sub.p R.sub.9 where p is 1, 1
or 2 and R.sub.9 is hydroxy, --ONa, or optionally substituted alkyl
of 1-12 carbons or optionally substituted cycloalkyl, or the
nontoxic, pharmaceutically acceptable salts thereof provided that
R.sub.1 and R.sub.4 are not both hydrogen and provided further that
R.sub.3 is not haloalkyl when R.sub.4 is hydrogen and R.sub.3 is
not alkyl when R.sub.4 is nitro.
2. A compound according to claim 1 wherein one of R.sub.1 and
R.sub.2 is hydrogen and the other is alkyl of 1-6 carbon atoms;
R.sub.3 is hydrogen, lower alkyl or cycloalkyl, lower alkoxy,
phenyl, the atoms necessary to complete a ring with adjacent carbon
atoms of the phenyl ring; --NR.sub.6 R.sub.7 where R.sub.6 is
hydrogen and R.sub.7 is --C(O)R.sub.8 where R.sub.8 is phenyl or
R.sub.6 and R.sub.7 together represent --(CH.sub.2).sub.x -- where
x is 2-6.
3. A compound according to claim 2 wherein R.sub.4 is --S(O).sub.p
R.sub.9 where p is 0, 1 or 2 and R.sub.9 is optionally substituted
alkyl of 1-12 carbons.
4. A compound according to claim 3 wherein R.sub.4 is --ZR.sub.5
where R.sub.5 is a lower alkyl carboxylic acid group.
5. A compound according to claim 4 wherein R.sub.4 is --SCH.sub.2
C(CH.sub.3).sub.2 COOH, --S(O)CH.sub.2 C(CH.sub.3).sub.2 CO.sub.2 H
or --SO.sub.2 CH.sub.2 C(CH.sub.3)CO.sub.2 H.
6. A compound according to claim 1 wherein R.sub.4 is --SCH.sub.3
in the ortho or para position.
7. A compound according to claim 5 wherein R.sub.4 is in the para
position.
8. A compound according to claim 1 wherein R.sub.1 and R.sub.2 are
different so as to be chiral.
9. A compound according to claim 1 wherein R.sub.1 and R.sub.2 are
both methyl or ethyl or together form a cycloalkyl ring.
10. A pharmaceutical composition for inhibiting undesired elastase
activity comprising an effective amount of a compound according to
claim 1 and a carrier therefor.
11. A method of inhibiting elastase activity which comprises
administering to a subject in need of such inhibition, an effective
amount of a compound of the formula: ##STR287## wherein: R.sub.1
and R.sub.2, which may be the same or different, are selected from
the group consisting of hydrogen, alkyl of 1-6 carbons, cycloalkyl
of 3-6 carbons or together represent a methylene group
--(CH.sub.2).sub.n -- where n is a whole number of from 1 to 6;
R.sub.3 represents one or more substituents up to five selected
from the group consisting of:
hydrogen, halogen, haloalkyl of 1-12 carbons, alkyl of 1-12
carbons, alkoxy of 1-12 carbons, alkenyl of 2-12 carbons,
cycloalkyl of 3-12 carbons, mono- or dicyclic aryl,
--ZR.sub.5 where Z is O, S, S(O) or SO.sub.2 and R.sub.5 is
hydrogen, alkyl of 1-18 carbons, cycloalkyl of 3-12 carbons or
phenyl;
--NR.sub.6 R.sub.7 where R.sub.6 and R.sub.7 may be the same or
different and may be hydrogen, alkyl of 1-12 carbons, cycloalkyl or
3-6 carbons, phenyl, alkoxy of 1-12 carbons, acyl of the formula
--C(O)R.sub.8 is alkyl of 1-12 carbons, cycloalkyl of 3-12 carbons,
phenyl, CH.sub.3 OC(O)CH.sub.2 CH.sub.2 --, HOOCCH.sub.2 CH.sub.2
--,
NaO.sub.3 SCH.sub.2 CH.sub.2 NHC(O)CH.sub.2 CH.sub.2 --, or R.sub.6
and R.sub.7 together may represent --C(O)CH.sub.2 CH.sub.2 C(O)--,
--C(O)--C.sub.6 H.sub.4 --C(O)-- or --(CH.sub.2).sub.x -- where x
is 2, 3, 4, 5 or 6;
morpholino, imidazole or piperazino joined to the phenyl ring
through a nitrogen atom; or
R.sub.1 represents an alkylene group containing 3 to 4 carbons, or
such group substituted with lower alkoxy or the group
--O(CH.sub.2).sub.n O-- where n is 2, 3 or 4, joining adjacent
carbons or the phenyl ring;
R.sub.4 is from one to five substituents selected from hydrogen,
halogen, nitro, --C(O)CH.sub.3, S(O).sub.p R.sub.9 where p is 1, 1
or 2 and R.sub.9 is hydroxy, --ONa, or optionally substituted alkyl
of 1-12 carbons or optionally substituted cycloalkyl, or the
nontoxic, pharmaceutically acceptable salts thereof provided that
R.sub.3 and R.sub.4 are not both hydrogen and provided further that
R.sub.3 is not haloalkyl when R.sub.4 is hydrogen and R.sub.3 is
not alkyl when R.sub.4 is nitro.
12. A compound according to claim 1 wherein R.sub.1 and R.sub.2 are
selected from the group consisting of hydrogen, alkyl of 1-6
carbons, cycloalkyl of 3-6 carbons or together represent a
methylene group --(CH.sub.2).sub.n -- where n is a whole number of
from 1 to 6;
R.sub.3 is alkyl in both the 3- and 4-positions or a polymethylene
group joining the 3- and 4-positions; and
R.sub.4 is --S(O).sub.p R.sub.9 where p is 0, 1 or 2 and R.sub.9 is
alkyl or carboxy-substituted alkyl of 1-12 carbons.
13. A compound according to claim 12 wherein R.sub.1 and R.sub.2
together represent a methylene group --(CH.sub.2).sub.n -- where n
is a whole number of from 1 to 6 and R.sub.3 is a
--(CH.sub.2).sub.4 -- group joining the 3- and 4-positions of the
ring.
14. A compound according to claim 13 wherein R.sub.4 is
--S(O).sub.p R.sub.9 where p is 0 and R.sub.9 is a butyl group
substituted with carboxy COOH.
15. A compound according to claim 14 wherein n is 3, p is 0,
R.sub.9 is --CH.sub.2 C(CH.sub.3).sub.2 COOH and the R.sub.4 is in
the 4-position of the ring.
Description
The present invention relates to certain 2-phenylalkanoate esters
which are useful as inhibitors of human leukocyte elastase (HLE) or
equivalently human neutrophil elastase (HNE).
BACKGROUND OF THE INVENTION
There has been considerable research effort in recent years toward
the development of HLE inhibitors because it appears that HLE may
be responsible for a variety of human diseases. For example, tests
have shown that there is an apparent association between HLE and
emphysema in Sandberg et al., The New England Journal of Medicine,
304:566 (1981). Other diseases and medical problems, such as
arthritis and related inflammatory conditions and dermatitis, have
also been associated with HLE. Accordingly, there is a need for
compounds which are effective to inhibit HLE.
Typical prior efforts to deal with elastase inhibition are
disclosed in the patent literature, for instance, U.S. Pat. Nos.
4,683,241 and 4,801,610.
SUMMARY OF THE INVENTION
The principal object of the present invention is to provide certain
new compounds which are useful as elastase inhibitors. These
compounds are characterized by their relatively low molecular
weight and high selectivity with respect to HLE. As a consequence,
they can be used to prevent, alleviate or otherwise treat disease
characterized by the degradation effects caused by HLE on
connective tissues in mammals, including humans.
The compounds of the invention may be structurally illustrated by
the following formula (VI): ##STR1## wherein:
R.sub.1 and R.sub.2, which may be the same or different, are
selected from the group consisting of:
hydrogen, alkyl of 1-6 carbons, cycloalkyl of 3 to 6 carbons or
together represent a methylene group --(CH.sub.2).sub.n -- where n
is a whole number of from 1 to 6;
R.sub.3 represents one or more substituents up to five selected
from the group consisting of:
hydrogen, halogen, haloalkyl of 1-12 carbons (e.g. CF.sub.3), alkyl
of 1-12 carbons, alkoxy of 1-12 carbons, cycloalkyl of 3-12
carbons, alkenyl of 2 to 12 carbons, mono- or dicyclic aryl (e.g.
optionally substituted phenyl or naphthyl),
--ZR.sub.5 where Z is O, S, S(O) or SO.sub.2 and R.sub.5 is
hydrogen alkyl of 1-18 carbons, cycloalkyl of 3-12 carbons or
phenyl;
--NR.sub.6 R.sub.7 where R.sub.6 and R.sub.7 may be the same or
different and may be hydrogen, alkyl of 1-12 carbons, cycloalkyl of
3-6 carbons, phenyl, alkoxy of 1-12 carbons, acyl of the formula
--C(O)R.sub.8 where R.sub.8 is alkyl of 1-12 carbons, cycloalkyl of
3-12 carbons, phenyl, CH.sub.3 OC(O)CH.sub.2 CH.sub.2 --,
HOOCCH.sub.2 CH.sub.2 --, NaO.sub.3 SCH.sub.2 CH.sub.2
NHC(O)CH.sub.2 CH.sub.2 --, or R.sub.6 and R.sub.7 together
represent --C(O)CH.sub.2 CH.sub.2 C(O)--, --C(O)--C.sub.6 H.sub.4
--C(O)-- or --(CH).sub.2).sub.x -- where x is 2, 3, 4, 5 or 6;
morpholino, imidazole or piperazino joined to the phenyl ring
through a nitrogen atom; or
R.sub.3 represents the atoms necessary to complete between adjacent
ring carbons a further carbocyclic ring of from 1 to 6 carbons or a
5-6 membered heterocyclic ring including one or more O, S or N ring
atoms; and
R.sub.4 is from one to five substituents selected from hydrogen,
halogen, nitro, --C(O)CH.sub.3, S(O).sub.p R.sub.9 where p is 0, 1
or 2 and R.sub.9 is hydroxy, --ONa or optionally substituted alkyl
of 1-12 carbons or optionally substituted cycloalkyl including, for
example, lower alkyl substituted with halogen (such as
trifluoromethyl) or lower alkyl bearing a carboxylic acid group,
especially --CH.sub.2 C(CH.sub.3).sub.2 CO.sub.2 H.
According to the invention, the phenyl rings may be unsubstituted
(i.e. R.sub.3 and R.sub.4 may both be hydrogen). However, it is
preferred that at least R.sub.4 be other than hydrogen.
It will be appreciated that when R.sub.1 and R.sub.2 are different,
the carbon atom to which these substituents are attached (i.e. the
"alpha carbon") is a chiral center and the resulting compounds may
exist in enantiomerically pure form or as racemic mixtures of the
enantiomers. The invention contemplates such mixtures (+/-) as well
as the separate (+ or -) enantiomers thereof.
Non-toxic pharmaceutically acceptable salts of the indicated
compounds are also contemplated.
PREFERRED EMBODIMENTS OF THE INVENTION
Particularly advantageous for present purposes are the compounds of
formula (VI) where one of R.sub.1 and R.sub.2 is hydrogen and the
other is alkyl, particularly ethyl; and R.sub.3 is hydrogen, lower
alkyl, cycloalkyl, lower alkoxy, phenyl, the atoms necessary to
complete an optionally substituted ring with the adjacent phenyl
group, piperidino or --NR.sub.6 R.sub.7 where R.sub.6 is hydrogen
and R.sub.7 is --C(O)R.sub.8 where R.sub.8 is phenyl or where
R.sub.6 and R.sub.7 together represent --(CH.sub.2).sub.x -- where
x is 2-6. The optional substitution in the case of R.sub.3 may be,
for example, lower alkyl or lower alkoxy, it being understood that
reference herein to lower alkyl or lower alkoxy contemplates up to
6 carbon atoms.
As a further feature of the invention, it has been found that
compounds which have been modified so as to remove the chiral
center at the alpha carbon, i.e. by making R.sub.1 and R.sub.2 the
same, e.g. either methyl or ethyl, or by merging R.sub.1 and
R.sub.2 into a cycloalkyl ring (such as cyclopropyl, cyclobutyl,
cyclopentyl or cyclohexyl) are particularly advantageous for use as
human neutrophil elastase inhibitors.
According to a further aspect of the invention, it has been found
that compounds wherein R.sub.4 is --SCH.sub.3 in the ortho or para
positions, or where R.sub.4 is --S--CH.sub.2 C(CH.sub.3).sub.2 COOH
in the para position, are particularly useful. These compounds
appear to be oxidatively activatable as in vivo inhibitors, i.e.
the --S-- (sulfide) group seems to be oxidized in situ to the
sulfoxide --S(O)-- or to the sulfone --S(O).sub.2 --. In this
regard, it has been found that the potency of the compounds where
R.sub.4 is --S-- (sulfide), --S(O)-- (sulfoxide) and --S(O).sub.2
-- (sulfone) increases in the series as follows:
Consequently, it appears that the potency of the --S-- compounds
can be increased by oxidants present at the site of HLE mediated
damage to form the corresponding sulfoxides or sulfones.
Representative compounds according to the invention are shown in
the following Tables I and II.
TABLE I
__________________________________________________________________________
This table exemplifies compounds of the formula (VI) wherein
R.sub.1 -R.sub.4 have the values indicated. Positioning of the
substituents represented by R.sub.3 and/or R.sub.4 is specified
numerically, the link of the ring to the rest of the molecule being
the 1-postion. Where two or more substituents are involved, each
position is identified. Compound No. R.sub.1 R.sub.2 R.sub.3
R.sub.4
__________________________________________________________________________
1) H C.sub.2 H.sub.5 H 4-SCH.sub.3 2) H C.sub.2 H.sub.5 H ##STR2##
3) H C.sub.2 H.sub.5 H ##STR3## 4) H C.sub.2 H.sub.5 H 4-NO.sub.2
5) H C.sub.2 H.sub.5 H 2-SCH.sub.3 6) H C.sub.2 H.sub.5 H ##STR4##
7) H C.sub.2 H.sub.5 H ##STR5## 8) H C.sub.2 H.sub.5 H 3-F,
4-NO.sub. 2 9) H C.sub.2 H.sub.5 H 4-NO.sub.2 10) H C.sub.2 H.sub.5
H 2,4-NO.sub.2 11) H C.sub.2 H.sub.5 H 2-NO.sub.2 12) H C.sub.2
H.sub.5 H 3-NO.sub.2 13) H C.sub.2 H.sub.5 H 4-F 14) H C.sub.2
H.sub.5 H 2,3,4-F 15) H C.sub.2 H.sub.5 H 3,4,5-F 16) H C.sub.2
H.sub.5 H 2,6-F 17) H C.sub.2 H.sub.5 H 2,3,5,6-F 18) H C.sub.2
H.sub.5 H 4-SO.sub.3 Na 19) H C.sub.2 H.sub.5 H ##STR6## 20) H
C.sub.2 H.sub.5 H ##STR7## 21) H C.sub.2 H.sub.5 4-OCH.sub.3
3-CH.sub.3, 4-SCH.sub.3 22) H C.sub.2 H.sub.5 4-OCH.sub.3 ##STR8##
23) H C.sub.2 H.sub.5 4-OCH.sub.3 ##STR9## 24) H C.sub.2 H.sub.5
4-OCH.sub.3 3-CH.sub.3, 4-NO.sub. 2 25) H C.sub.2 H.sub.5
4-OCH.sub.3 2-CH.sub.3, 4-SCH.sub.3 26) H C.sub.2 H.sub.5
4-OCH.sub.3 ##STR10## 27) H C.sub.2 H.sub.5 4-OCH.sub.3 ##STR11##
28) H C.sub.2 H.sub.5 4-OCH.sub.3 2CH.sub.3, 4-NO.sub.2 29) H
C.sub.2 H.sub.5 4-OCH.sub.3 2,6-CH.sub.3, 4-SCH.sub.3 30) H C.sub.2
H.sub.5 4-OCH.sub.3 ##STR12## 31) H C.sub.2 H.sub.5 4-OCH.sub.3
##STR13## 32) H C.sub.2 H.sub.5 4-OCH.sub.3 2,6-CH.sub.3,
4-NO.sub.2 33) H C.sub.2 H.sub.5 H 2,3,4,5,6-F 34) H C.sub.2
H.sub.5 4-OCH.sub.3 4-NO.sub.2 35) H C.sub.2 H.sub.5 4-OCH.sub.3
4-SCH.sub.3 36) H C.sub.2 H.sub.5 4-OCH.sub.3 ##STR14## 37) H
C.sub.2 H.sub.5 4-OCH.sub. 3 ##STR15## 38) H C.sub.2 H.sub.5
4-OC.sub.2 H.sub.5 4-SCH.sub.3 39) H C.sub.2 H.sub.5 4-OC.sub.2
H.sub.5 ##STR16## 40) H C.sub.2 H.sub.5 4-OC.sub.2 H.sub.5
##STR17## 41) H C.sub.2 H.sub.5 4-OC.sub.2 H.sub.5 4-NO.sub.2 42) H
C.sub.2 H.sub.5 4-OC.sub.4 H.sub.9 4-SCH.sub.3 43) H C.sub.2
H.sub.5 4-OC.sub.4 H.sub.9 ##STR18## 44) H C.sub.2 H.sub.5
4-OC.sub.4 H.sub.9 ##STR19## 45) H C.sub.2 H.sub.5 3,4,5-OCH.sub.3
4-SCH.sub.3 46) H C.sub.2 H.sub.5 3,4,5-OCH.sub.3 ##STR20## 47) H
C.sub.2 H.sub.5 3,4,5-OCH.sub.3 ##STR21## 48) H C.sub.2 H.sub.5
3,4-OCH.sub.3 4-SCH.sub.3 49) H C.sub.2 H.sub.5 3,4-OCH.sub. 3
##STR22## 50) H C.sub.2 H.sub.5 3,4-OCH.sub.3 ##STR23## 51) H
C.sub.2 H.sub.5 3,4-OCH.sub.3 4-NO.sub.2 52) H C.sub.2 H.sub.5 4-OH
4-SCH.sub.3 53) H C.sub.2 H.sub.5 4-OH ##STR24## 54) H C.sub.2
H.sub.5 4-OH ##STR25## 55) H C.sub.2 H.sub.5 4-OH 4-NO.sub.2 56) H
C.sub.2 H.sub.5 3-OH 4-SCH.sub.3 57) H C.sub.2 H.sub.5 3-OH
##STR26## 58) H C.sub.2 H.sub.5 3-OH ##STR27## 59) H C.sub.2
H.sub.5 3-OH 4-NO.sub.2 60) H C.sub.2 H.sub.5 3-OCH.sub.3
4-SCH.sub.3 61) H C.sub.2 H.sub.5 3-OCH.sub.3 ##STR28## 62) H
C.sub.2 H.sub.5 3-OCH.sub.3 ##STR29## 63) H C.sub.2 H.sub.5
3-OCH.sub.3 4-NO.sub.2 64) H C.sub.2 H.sub.5 4-OC.sub.9 H.sub.19
4-SCH.sub.3 65) H C.sub.2 H.sub.5 4-OC.sub.9 H.sub.19 ##STR30## 66)
H C.sub.2 H.sub.5 4-OC.sub.9 H.sub.19 ##STR31## 67) H C.sub.2
H.sub.5 4-OC.sub.9 H.sub.19 4-NO.sub.2 68) H C.sub.2 H.sub.5
4-OCH.sub.2 CO.sub.2 C.sub.2 H.sub.5 4-SCH.sub.3 69) H C.sub.2
H.sub.5 4-OCH.sub.2 CO.sub.2 C.sub.2 H.sub.5 ##STR32## 70) H
C.sub.2 H.sub.5 4-OCH.sub.2 CO.sub.2 C.sub.2 H.sub.5 ##STR33## 71)
H C.sub.2 H.sub.5 4-OCH.sub.2 CO.sub.2 C.sub.2 H.sub.5 4-NO.sub.2
72) H n-C.sub.3 H.sub.7 4-OCH.sub.3 4-SCH.sub.3 73) H n-C.sub.3
H.sub.7 4-OCH.sub.3 ##STR34## 74) H n-C.sub.3 H.sub.7 4-OCH.sub.3
##STR35## 75 H n-C.sub. 3 H.sub.7 4-OCH.sub.3 4-NO.sub.2 76) H
n-C.sub.4 H.sub.9 4-OCH.sub.3 4-SCH.sub.3 77) H n-C.sub.4 H.sub.9
4-OCH.sub.3 ##STR36## 78) H n-C.sub.4 H.sub.9 4-OCH.sub.3 ##STR37##
79) H n-C.sub.4 H.sub.9 4-OCH.sub.3 4-NO.sub.2 80) H C.sub.2
H.sub.5 3-OCH.sub.3, 4-SCH.sub.3 4-OC.sub.2 H.sub.5 81) H C.sub.2
H.sub.5 3-OCH.sub.3, 4-OC.sub.2 H.sub.5 ##STR38## 82) H C.sub.2
H.sub.5 3-OCH.sub.3, 4-OC.sub.2 H.sub.5 ##STR39## 83) H C.sub.2
H.sub.5 3-OCH.sub.3, 4-NO.sub.2 4-OC.sub.2 H.sub.5 84) H C.sub.2
H.sub.5 3,5-OCH.sub.3 4-SCH.sub.3 85) H C.sub.2 H.sub.5
3,5-OCH.sub.3 ##STR40## 86) H C.sub.2 H.sub.5 3,5-OCH.sub.3
##STR41## 87) H C.sub.2 H.sub.5 3,5-OCH.sub.3 4-NO.sub.2 88) H
C.sub.2 H.sub.5 3-OC.sub.2 H.sub.5, 4-SCH.sub.3 4-OCH.sub.3 89) H
C.sub.2 H.sub.5 3-OC.sub.2 H.sub.5, 4-OCH.sub.3 ##STR42## 90) H
C.sub.2 H.sub.5 3-OC.sub.2 H.sub.5, 4-OCH.sub.3 ##STR43## 91) H
C.sub.2 H.sub.5 3-OC.sub.2 H.sub.5, 4-NO.sub.2 4-OCH.sub.3
92) H C.sub.2 H.sub.5 4-OC.sub.6 H.sub.5 4-SCH.sub.3 93) H C.sub.2
H.sub.5 4-OC.sub.6 H.sub.5 ##STR44## 94) H C.sub.2 H.sub.5
4-OC.sub.6 H.sub.5 ##STR45## 95) H C.sub.2 H.sub.5 4-OC.sub.6
H.sub.5 4-NO.sub.2 96) H C.sub.2 H.sub.5 3-OC.sub.6 H.sub.5
4-SCH.sub.3 97) H C.sub.2 H.sub.5 3-OC.sub.6 H.sub.5 ##STR46## 98)
H C.sub.2 H.sub.5 3-OC.sub.6 H.sub.5 ##STR47## 99) H C.sub.2
H.sub.5 3-OC.sub.6 H.sub.5 4-NO.sub.2 100) H C.sub.2 H.sub.5
4-CH.sub.3 4-SCH.sub.3 101) H C.sub.2 H.sub.5 4-CH.sub.3 ##STR48##
102) H C.sub.2 H.sub.5 4-CH.sub.3 ##STR49## 103) H C.sub.2 H.sub.5
4-CH.sub.3 4-NO.sub.2 104) H C.sub.2 H.sub.5 3-CH.sub.3 4-SCH.sub.3
105) H C.sub.2 H.sub.5 3-CH.sub.3 ##STR50## 106) H C.sub.2 H.sub.5
3-CH.sub.3 ##STR51## 107) H C.sub.2 H.sub.5 3-CH.sub.3 4-NO.sub.2
108) H C.sub.2 H.sub.5 3,4-CH.sub.3 4-SCH.sub.3 109) H C.sub.2
H.sub.5 3,4-CH.sub.3 ##STR52## 110) H C.sub.2 H.sub.5 3,4-CH.sub.3
##STR53## 111) H C.sub.2 H.sub.5 3,4-CH.sub.3 4-NO.sub.2 112) H
C.sub.2 H.sub.5 3,4-C.sub.2 H.sub.5 4-SCH.sub.3 113) H C.sub.2
H.sub.5 3,4-C.sub.2 H.sub.5 ##STR54## 114) H C.sub.2 H.sub.5
3,4-C.sub.2 H.sub.5 ##STR55## 115) H C.sub.2 H.sub.5 3,4-C.sub.2
H.sub.5 4-NO.sub.2 116) H C.sub.2 H.sub.5 4-C.sub.2 H.sub.5
4-SCH.sub.3 117) H C.sub.2 H.sub.5 4-C.sub.2 H.sub.5 ##STR56## 118)
H C.sub.2 H.sub.5 4-C.sub.2 H.sub.5 ##STR57## 119) H C.sub.2
H.sub.5 4-C.sub.2 H.sub.5 4-NO.sub.2 120) H C.sub.2 H.sub.5
4-C.sub.3 H.sub.7 4-SCH.sub.3 121) H C.sub.2 H.sub.5 4-C.sub.3
H.sub.7 ##STR58## 122) H C.sub.2 H.sub.5 4-C.sub.3 H.sub.7
##STR59## 123) H C.sub.2 H.sub.5 4-C.sub.3 H.sub.7 4-NO.sub.2 124)
H C.sub.2 H.sub.5 4-CH(CH.sub.3).sub.2 4-SCH.sub. 3 125) H C.sub.2
H.sub.5 4-CH(CH.sub.3).sub.2 ##STR60## 126) H C.sub.2 H.sub.5
4-CH(CH.sub.3).sub.2 ##STR61## 127) H C.sub.2 H.sub.5
4-CH(CH.sub.3).sub.2 4-NO.sub.2 128) H C.sub.2 H.sub.5 4-n-C.sub.4
H.sub.9 4-SCH.sub.3 129) H C.sub.2 H.sub.5 4-n-C.sub.4 H.sub.9
##STR62## 130) H C.sub.2 H.sub.5 4-n-C.sub.4 H.sub.9 ##STR63## 131)
H C.sub.2 H.sub.5 4-n-C.sub.4 H.sub.9 4-NO.sub.2 132) H CH.sub.3
4-CH.sub.2 CH(CH.sub.3).sub.2 4-SCH.sub.3 133) H CH.sub.3
4-CH.sub.2 CH(CH.sub.3).sub.2 ##STR64## 134) H CH.sub.3 4-CH.sub.2
CH(CH.sub.3).sub.2 ##STR65## 135) H C.sub.2 H.sub.5 4-CH.sub.2
CH(CH.sub.3).sub.2 4-SCH.sub.3 136) H C.sub.2 H.sub.5 4-CH.sub.2
CH(CH.sub.3).sub.2 ##STR66## 137) H C.sub.2 H.sub.5 4-CH.sub.2
CH(CH.sub.3).sub.2 4-NO.sub.2 138) H C.sub.2 H.sub.5 4-CH.sub.2
CH(CH.sub.3).sub.2 ##STR67## 139) H C.sub.2 H.sub.5
4-C(CH.sub.3).sub.3 4-SCH.sub.3 140) H C.sub.2 H.sub.5
4-C(CH.sub.3).sub.3 ##STR68## 141) H C.sub.2 H.sub.5
4-C(CH.sub.3).sub.3 ##STR69## 142) H C.sub.2 H.sub.5
4-C(CH.sub.3).sub.3 4-NO.sub.2 143) H C.sub.2 H.sub.5 4-n-C.sub.5
H.sub.11 4-SCH.sub.3 144) H C.sub.2 H.sub.5 4-n-C.sub.5 H.sub.11
##STR70## 145) H C.sub.2 H.sub.5 4-n-C.sub.5 H.sub.11 ##STR71##
146) H C.sub.2 H.sub.5 4-n-C.sub.5 H.sub.11 4-NO.sub.2 147) H
C.sub.2 H.sub.5 4-cyclohexyl 4-SCH.sub.3 148) H C.sub.2 H.sub.5
4-cyclohexyl ##STR72## 149) H C.sub.2 H.sub.5 4-cyclohexyl
##STR73## 150) H C.sub.2 H.sub.5 4-cyclohexyl 4-NO.sub.2 151) H
C.sub.2 H.sub.5 4-cyclopropyl 4-SCH.sub.3 152) H C.sub.2 H.sub.5
4-cyclopropyl ##STR74## 153) H C.sub.2 H.sub.5 4-cyclopropyl
##STR75## 154) H C.sub.2 H.sub.5 4-cyclopropyl 4-NO.sub.2 155) H
C.sub.2 H.sub.5 4-SCH.sub.3 4-SCH.sub.3 156) H C.sub.2 H.sub.5
4-SCH.sub.3 ##STR76## 157) H C.sub.2 H.sub.5 4-SCH.sub.3 ##STR77##
158) H C.sub.2 H.sub.5 4-SCH.sub.3 4-NO.sub.2 159) H C.sub.2
H.sub.5 4-SC.sub.2 H.sub.5 4-SCH.sub.3 160) H C.sub.2 H.sub.5
4-SC.sub.2 H.sub.5 ##STR78## 161) H C.sub.2 H.sub.5 4-SC.sub.2
H.sub.5 ##STR79## 162) H C.sub.2 H.sub.5 4-SC.sub.2 H.sub.5
4-NO.sub.2 163) H C.sub.2 H.sub.5 ##STR80## 4-SCH.sub.3 164) H
C.sub.2 H.sub.5 ##STR81## ##STR82## 165) H C.sub.2 H.sub.5
##STR83## ##STR84## 166) H C.sub.2 H.sub.5 4-N(CH.sub.3).sub.2
4-SCH.sub.3 167) H C.sub.2 H.sub.5 4-N(CH.sub.3).sub.2 ##STR85##
168) H C.sub.2 H.sub.5 4-N(CH.sub.3).sub.2 ##STR86## 169) H C.sub.2
H.sub.5 4-N(CH.sub.3).sub.2 4-NO.sub.2 170) H C.sub.2 H.sub.5
4-N(C.sub.2 H.sub.5).sub.2 4-SCH.sub.3 171) H C.sub.2 H.sub.5
4-N(C.sub.2 H.sub.5).sub.2 ##STR87## 172) H C.sub.2 H.sub.5
4-N(C.sub.2 H.sub.5).sub.2 ##STR88## 173) H C.sub.2 H.sub.5
4-N(C.sub.2 H.sub.5).sub.2 4-NO.sub.2 174) H C.sub.2 H.sub.5
3-N(CH.sub.3).sub.2 4-SCH.sub.3 175) H C.sub.2 H.sub.5
3-N(CH.sub.3).sub.2 ##STR89## 176) H C.sub.2 H.sub.5
3-N(CH.sub.3).sub.2 ##STR90## 177) H C.sub.2 H.sub.5
3-N(CH.sub.3).sub.2 4-NO.sub.2 178) H C.sub.2 H.sub.5 4-N.sub.3
4-NO.sub.2 179) H C.sub.2 H.sub.5 ##STR91## 4-SCH.sub.3 180) H
C.sub.2 H.sub.5 ##STR92## ##STR93## 181) H C.sub.2 H.sub.5
##STR94## ##STR95##
182) H C.sub.2 H.sub.5 ##STR96## 4-NO.sub.2 183) H C.sub.2 H.sub.5
##STR97## 4-SCH.sub.3 184 H C.sub.2 H.sub.5 ##STR98## ##STR99##
185) H C.sub.2 H.sub.5 ##STR100## ##STR101## 186) H C.sub.2 H.sub.5
##STR102## 4-NO.sub.2 187) H C.sub.2 H.sub.5 ##STR103## 4-SCH.sub.3
188) H C.sub.2 H.sub.5 ##STR104## ##STR105## 189) H C.sub.2 H.sub.5
##STR106## ##STR107## 190) H C.sub.2 H.sub.5 ##STR108## 4-NO.sub.2
191) H C.sub.2 H.sub.5 ##STR109## 4-SCH.sub.3 192) H C.sub.2
H.sub.5 ##STR110## ##STR111## 193) H C.sub.2 H.sub.5 ##STR112##
##STR113## 194) H C.sub.2 H.sub.5 ##STR114## 4-NO.sub.2 195) H
C.sub.2 H.sub.5 ##STR115## 4-SCH.sub.3 196) H C.sub.2 H.sub.5
##STR116## ##STR117## 197) H C.sub.2 H.sub.5 ##STR118## ##STR119##
198) H C.sub.2 H.sub.5 ##STR120## 4-NO.sub.2 199) H C.sub.2 H.sub.5
4-F 4-SCH.sub.3 200) H C.sub.2 H.sub.5 4-F ##STR121## 201) H
C.sub.2 H.sub.5 4-F ##STR122## 202) H C.sub.2 H.sub.5 4-F
4-NO.sub.2 203) H C.sub.2 H.sub.5 3-F 4-SCH.sub.3 204) H C.sub.2
H.sub.5 3-F ##STR123## 205) H C.sub.2 H.sub.5 3-F ##STR124## 206) H
C.sub.2 H.sub.5 3-F 4-NO.sub.2 207) H C.sub.2 H.sub.5 4-Cl
4-SCH.sub.3 208) H C.sub.2 H.sub.5 4-Cl ##STR125## 209) H C.sub.2
H.sub.5 4-Cl ##STR126## 210) H C.sub.2 H.sub.5 4-Cl 4-NO.sub.2 211)
H C.sub.2 H.sub.5 3-Cl 4-SCH.sub.3 212) H C.sub.2 H.sub.5 3-Cl
##STR127## 213) H C.sub.2 H.sub.5 3-Cl ##STR128## 214) H C.sub.2
H.sub.5 3-Cl 4-NO.sub.2 215) H C.sub.2 H.sub.5 4-Br 4-SCH.sub.3
216) H C.sub.2 H.sub.5 4-Br ##STR129## 217) H C.sub.2 H.sub.5 4-Br
##STR130## 218) H C.sub.2 H.sub.5 4-Br 4-NO.sub.2 219) H C.sub.2
H.sub.5 3-Br 4-SCH.sub.3 220) H C.sub.2 H.sub.5 3-Br ##STR131##
221) H C.sub.2 H.sub.5 3-Br ##STR132## 222) H C.sub.2 H.sub.5 3-Br
4-NO.sub.2 223) H C.sub.2 H.sub.5 4-NHCOC.sub.6 H.sub.5 4-SCH.sub.3
224) H C.sub.2 H.sub.5 4-NHCOC.sub.6 H.sub.5 ##STR133## 225) H
C.sub.2 H.sub.5 4-NHCOC.sub.6 H.sub.5 ##STR134## 226) H C.sub.2
H.sub.5 4-NHCOC.sub.6 H.sub.5 4-NO.sub.2 227) H C.sub.2 H.sub.5
##STR135## 4-SCH.sub.3 228) H C.sub.2 H.sub.5 ##STR136## ##STR137##
229) H C.sub.2 H.sub.5 ##STR138## ##STR139## 230) H C.sub.2 H.sub.5
##STR140## 4-NO.sub.2 231) H C.sub.2 H.sub.5 ##STR141## 4-SCH.sub.3
232) H C.sub.2 H.sub.5 ##STR142## ##STR143## 233) H C.sub.2 H.sub.5
##STR144## ##STR145## 234) H C.sub.2 H.sub.5 ##STR146## 4-SCH.sub.3
235) H C.sub.2 H.sub.5 ##STR147## ##STR148## 236) H C.sub.2 H.sub.5
##STR149## ##STR150## 237) H C.sub.2 H.sub.5 ##STR151## 4-SCH.sub.3
238) H C.sub.2 H.sub.5 ##STR152## ##STR153## 239) H C.sub.2 H.sub.5
##STR154## ##STR155## 240 H C.sub.2 H.sub.5 4-NH.sub.2 4-SCH.sub.3
241) H C.sub.2 H.sub.5 4-NH.sub.2 ##STR156## 242) H C.sub.2 H.sub.5
4-NH.sub.2 ##STR157## 243) H C.sub.2 H.sub.5 4-NH.sub.2 4-NO.sub.2
244) H C.sub.2 H.sub.5 ##STR158## 4-SCH.sub.3 245) H C.sub.2
H.sub.5 ##STR159## ##STR160## 246) H C.sub.2 H.sub.5 ##STR161##
##STR162## 247) H C.sub.2 H.sub.5 ##STR163## 4-NO.sub.2 248) H
C.sub.2 H.sub.5 ##STR164## 4-SCH.sub.3 249) H C.sub.2 H.sub.5
##STR165## ##STR166## 250) H C.sub.2 H.sub.5 ##STR167## ##STR168##
251) H C.sub.2 H.sub.5 ##STR169## 4-NO.sub.2 252) H C.sub.2 H.sub.5
##STR170## 4-SCH.sub.3 253) H C.sub.2 H.sub.5 ##STR171## ##STR172##
254) H C.sub.2 H.sub.5 ##STR173## ##STR174## 255) H C.sub.2 H.sub.5
##STR175## 4-NO.sub.2 256) H C.sub.2 H.sub.5 ##STR176## 4-SCH.sub.3
257) H C.sub.2 H.sub.5 ##STR177## ##STR178## 258) H C.sub.2 H.sub.5
##STR179## ##STR180## 259) H C.sub.2 H.sub.5 ##STR181## 4-NO.sub.2
260) H C.sub.2 H.sub.5 ##STR182## 4-SCH.sub.3 261) H C.sub.2
H.sub.5 ##STR183## ##STR184## 262) H C.sub.2 H.sub.5 ##STR185##
##STR186## 263) H C.sub.2 H.sub.5 ##STR187## 4-NO.sub.2 264) H
C.sub.2 H.sub.5 4-C.sub.6 H.sub.5 4-SCH.sub.3 265) H C.sub. 2
H.sub.5 4-C.sub.6 H.sub.5 ##STR188## 266) H C.sub.2 H.sub.5
4-C.sub.6 H.sub.5 ##STR189## 267) H C.sub.2 H.sub.5 4-C.sub.6
H.sub.5 4-NO.sub.2 268) CH.sub.3 CH.sub.3 H 4-SCH.sub.3 269)
CH.sub.3 CH.sub.3 H ##STR190## 270) CH.sub.3 CH.sub.3 H ##STR191##
271) CH.sub.3 CH.sub.3 H 4-NO.sub.2 272) C.sub.2 H.sub.5 C.sub.2
H.sub.5 H 4-SCH.sub.3 273) C.sub.2 H.sub.5 C.sub.2 H.sub.5 H
##STR192## 274) C.sub.2 H.sub.5 C.sub.2 H.sub.5 H ##STR193## 275)
C.sub.2 H.sub.5 C.sub.2 H.sub.5 H 4-NO.sub.2 276) (CH.sub.2).sub.2
H 4-SCH.sub.3 277) (CH.sub.2).sub.2 H ##STR194##
278) (CH.sub.2).sub.2 H ##STR195## 279) (CH.sub.2).sub.2 H
4-NO.sub.2 280) (CH.sub.2).sub.3 H 4-SCH.sub.3 281)
(CH.sub.2).sub.3 H ##STR196## 282) (CH.sub.2).sub.3 H ##STR197##
283) (CH.sub.2).sub.3 H 4-NO.sub.2 284) (CH.sub.2).sub.4 H
4-SCH.sub.3 285) (CH.sub.2).sub.4 H ##STR198## 286)
(CH.sub.2).sub.4 H ##STR199## 287) (CH.sub.2).sub.4 H 4-NO.sub.2
288) CH.sub.3 CH.sub.3 4-OCH.sub.3 4-SCH.sub.3 289) CH.sub.3
CH.sub.3 4-OCH.sub.3 ##STR200## 290) CH.sub.3 CH.sub.3 4-OCH.sub.3
##STR201## 291) CH.sub.3 CH.sub.3 4-OCH.sub.3 4-NO.sub.2 292)
C.sub.2 H.sub.5 C.sub.2 H.sub.5 4-OCH.sub.3 4-SCH.sub.3 293)
C.sub.2 H.sub.5 C.sub.2 H.sub.5 4-OCH.sub.3 ##STR202## 294) C.sub.2
H.sub.5 C.sub.2 H.sub.5 4-OCH.sub.3 ##STR203## 295) C.sub.2 H.sub.5
C.sub.2 H.sub.5 4-OCH.sub.3 4-NO.sub.2 296) (CH.sub.2).sub.2
4-OCH.sub.3 4-SCH.sub.3 297) (CH.sub.2).sub.2 4-OCH.sub.3
##STR204## 298) (CH.sub.2).sub.2 4-OCH.sub.3 ##STR205## 299)
(CH.sub.2).sub.2 4-OCH.sub.3 4-NO.sub.2 300) (CH.sub.2).sub.3
4-OCH.sub.3 4-SCH.sub.3 301) (CH.sub.2).sub.3 4-OCH.sub.3
##STR206## 302) (CH.sub.2).sub.3 4-OCH.sub.3 ##STR207## 303)
(CH.sub.2).sub.3 4-OCH.sub.3 4-NO.sub.2 304) (CH.sub.2).sub.4
4-OCH.sub.3 4-SCH.sub.3 305) (CH.sub.2).sub.4 4-OCH.sub.3
##STR208## 306) (CH.sub.2).sub.4 4-OCH.sub.3 ##STR209## 307
(CH.sub.2).sub.4 4-OCH.sub.3 4-NO.sub.2 308) CH.sub.3 CH.sub.3
4-OCH.sub.3 4-SCH.sub.2 C(CH.sub. 3).sub.2 CO.sub.2 H 309) CH.sub.3
CH.sub.3 4-OCH.sub.3 ##STR210## 310) CH.sub.3 CH.sub.3 4-OCH.sub.3
##STR211## 311) (CH.sub.2).sub.3 4-OCH.sub.3 4-SCH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 312) (CH.sub.2).sub.3 4-OCH.sub.3
##STR212## 313) (CH.sub.2).sub.3
4-OCH.sub.3 ##STR213## 314) CH.sub.3 CH.sub.3 H 4-SCH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 315) CH.sub.3 CH.sub.3 H ##STR214##
316) CH.sub.3 CH.sub.3 H ##STR215## 317) (CH.sub.2).sub.3 H
4-SCH.sub.2 C(CH.sub.3).sub. 2 CO.sub.2 H 318) (CH.sub.2).sub.3 H
##STR216## 319) (CH.sub.2).sub.3 H ##STR217## 320) C.sub.2 H.sub.5
C.sub.2 H.sub.5 H 4-SCH.sub.2 C(CH.sub.3).sub.2 CO.sub.2 H 321)
C.sub.2 H.sub.5 C.sub.2 H.sub.5 H ##STR218## 322) C.sub.2 H.sub.5
C.sub.2 H.sub.5 H ##STR219## 323) H C.sub.2 H.sub.5 H 4-SCH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 324) H C.sub.2 H.sub.5 H ##STR220##
325) H C.sub.2 H.sub.5 H ##STR221## 326) H C.sub.2 H.sub.5
##STR222## 4-SCH.sub.2 C(CH.sub.3).sub.2 CO.sub.2 H 327) H C.sub.2
H.sub.5 ##STR223## ##STR224## 328) H C.sub.2 H.sub.5 ##STR225##
##STR226## 329) H C.sub.2 H.sub.5 4-cyclohexyl 4-SCH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 330) H C.sub.2 H.sub.5 4-cyclohexyl
##STR227## 331) H C.sub.2 H.sub.5 4-cyclohexyl ##STR228## 332) H
C.sub.2 H.sub.5 4-C.sub.2 H.sub.5 4-SCH.sub.2 C(CH.sub.3).sub.2
CO.sub.2 H 333) H C.sub.2 H.sub.5 4-C.sub.2 H.sub.5 ##STR229## 334)
H C.sub.2 H.sub.5 4-C.sub.2 H.sub.5 ##STR230## 335) H C.sub.2
H.sub.5 3,4-C.sub.2 H.sub.5 4-SCH.sub.2 C(CH.sub.3).sub.2 CO.sub.2
H 336) H C.sub.2 H.sub.5 3,4-C.sub.2 H.sub.5 ##STR231## 337) H
C.sub.2 H.sub.5 3,4-C.sub.2 H.sub.5 ##STR232## 338) H C.sub.2
H.sub.5 4-NHCOC.sub.6 H.sub.5 4-SCH.sub.2 C(CH.sub.3).sub.2
CO.sub.2 H 339) H C.sub.2 H.sub.5 4-NHCOC.sub.6 H.sub.5 ##STR233##
340) H C.sub.2 H.sub.5 4-NHCOC.sub.6 H.sub.5 ##STR234## 341)
CH.sub.3 CH.sub.3 3,4-C.sub.2 H.sub.5 4-SCH.sub.2 C(CH.sub.3).sub.2
CO.sub.2 H 342) CH.sub.3 CH.sub.3 3,4-C.sub.2 H.sub.5 ##STR235##
343) CH.sub.3 CH.sub.3 3,4-C.sub.2 H.sub. 5 ##STR236## 344)
(CH.sub.2).sub.3 3,4-C.sub.2 H.sub.5 4-SCH.sub.2 C(CH.sub.3).sub.2
CO.sub.2 H 345) (CH.sub.2).sub.3 3,4-C.sub.2 H.sub.5 ##STR237##
346) (CH.sub.2).sub.3 3,4-C.sub.2 H.sub.5 ##STR238## 347) CH.sub.3
CH.sub.3 4-cyclohexyl 4-SCH.sub.2 C(CH.sub.3).sub.2 CO.sub.2 H 348)
CH.sub.3 CH.sub.3 4-cyclohexyl ##STR239## 349) CH.sub.3 CH.sub.3
4-cyclohexyl ##STR240## 350) (CH.sub.2).sub.3 4-cyclohexyl
4-SCH.sub.2 C(CH.sub.3).sub.2 CO.sub.2 H 351) (CH.sub.2).sub.3
4-cyclohexyl ##STR241## 352) (CH.sub.2 ).sub.3 4-cyclohexyl
##STR242## 353) CH.sub.3 CH.sub.3 ##STR243## 4-SCH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 354) CH.sub.3 CH.sub.3 ##STR244##
##STR245## 355) CH.sub.3 CH.sub.3 ##STR246## ##STR247## 356)
(CH.sub.2).sub.3 ##STR248## 4-SCH.sub.2 C(CH.sub.3).sub.2 CO.sub.2
H 357) (CH.sub.2).sub.3 ##STR249## ##STR250## 358) (CH.sub.2).sub.3
##STR251## ##STR252##
__________________________________________________________________________
TABLE II
__________________________________________________________________________
Other compounds contemplated herein include the following compounds
of formula (X): ##STR253## (X) Compound No. R.sub.1 R.sub.2 R.sub.3
R.sub.4
__________________________________________________________________________
359) H C.sub.2 H.sub.5 3-(CH.sub.2).sub.3 -4 4-SCH.sub.3 360) H
C.sub.2 H.sub.5 3-(CH.sub.2).sub.3 -4 ##STR254## 361) H C.sub.2
H.sub.5 3-(CH.sub.2).sub.3 -4 ##STR255## 362) H C.sub.2 H.sub.5
3-(CH.sub.2).sub.3 -4 4-NO.sub.2 363) H C.sub.2 H.sub.5
3-(CH.sub.2).sub.4 -4 4-SCH.sub.3 364) H C.sub.2 H.sub.5
3-(CH.sub.2).sub.4 -4 ##STR256## 365) H C.sub.2 H.sub.5
3-(CH.sub.2).sub.4 -4 ##STR257## 366) H C.sub.2 H.sub.5
3-(CH.sub.2).sub.4 -4 4-NO.sub.2 367) H C.sub.2 H.sub.5 3-OCH.sub.2
CH.sub.2 O-4 4-SCH.sub.3 368) H C.sub.2 H.sub.5 3-OCH.sub.2
CH.sub.2 O-4 ##STR258## 369) H C.sub.2 H.sub.5 3-OCH.sub.2 CH.sub.2
O-4 ##STR259## 370) H C.sub.2 H.sub.5 3-OCH.sub.2 CH.sub.2 O-4
4-NO.sub.2 371) H C.sub.2 H.sub.5 3-OCH.sub.2 O-4 4-SCH.sub.3 372)
H C.sub.2 H.sub.5 3-OCH.sub.2 O-4 ##STR260## 373) H C.sub.2 H.sub.5
3-OCH.sub.2 O-4 ##STR261## 374) H C.sub.2 H.sub.5 3-OCH.sub.2 O-4
4-NO.sub.2 375) (+) H CH.sub.3 3-CHCHC(OCH.sub.3)CH-4 4-SCH.sub.3
376) (+) H CH.sub.3 3-CHCHC(OCH.sub.3)CH-4 ##STR262## 377) (+) H
CH.sub.3 3-CHCHC(OCH.sub.3)CH-4 ##STR263## 378) (+/-) H CH.sub.3
3-CHCHC(OCH.sub. 3)CH-4 4-SCH.sub.3 379) (+/-) H CH.sub.3
3-CHCHC(OCH.sub.3)CH-4 ##STR264## 380) (+/-) H CH.sub.3
3-CHCHC(OCH.sub.3)CH-4 ##STR265## 381) (+/-) H CH.sub.3
3-CHCHC(OCH.sub.3)CH-4 4-NO.sub.2 382) (+/-) H C.sub.2 H.sub.5
3-CHCHC(OCH.sub.3)CH-4 4-SCH.sub.3 383) (+/-) H C.sub.2 H.sub.5
3-CHCHC(OCH.sub.3)CH-4 ##STR266## 384) (+/-) H C.sub.2 H.sub.5
3-CHCHC(OCH.sub.3)CH-4 ##STR267## 385) (+/-) H C.sub.2 H.sub.5
3-CHCHC(OCH.sub.3)CH-4 4-NO.sub.2 386) (+/-) H C.sub.2 H.sub.5
3-CHCHC(OCH.sub.3)CH-4 4-SCH.sub.2 C(CH.sub.3).sub.2 CO.sub.2 H
387) (+/-) H C.sub.2 H.sub.5 3-CHCHC(OCH.sub.3)CH-4 ##STR268## 388)
(+/-) H C.sub.2 H.sub.5 3-CHCHC(OCH.sub.3)CH-4 ##STR269## 389) H
C.sub.2 H.sub.5 3-(CH.sub.2).sub.4 -4 4-SCH.sub.2 C(CH.sub.3).sub.2
CO.sub.2 H 390) H C.sub.2 H.sub.5 3-(CH.sub.2).sub.4 -4 ##STR270##
391) H C.sub.2 H.sub.5 3-(CH.sub.2).sub.4 -4 ##STR271## 392)
CH.sub.3 CH.sub.3 3-(CH.sub.2).sub.4 -4 4-SCH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 393) CH.sub.3 CH.sub.3
3-(CH.sub.2).sub.4 -4 ##STR272## 394) CH.sub.3 CH.sub.3
3-(CH.sub.2).sub.4 -4 ##STR273## 395) (CH.sub.2).sub.3
3-(CH.sub.2).sub.4 -4 4-SCH.sub.2 C(CH.sub.3).sub.2 CO.sub.2 H 396)
(CH.sub.2).sub.3 3-(CH.sub.2).sub.4 -4 ##STR274## 397)
(CH.sub.2).sub.3 3-(CH.sub.2).sub.4 -4 ##STR275##
__________________________________________________________________________
Broadly described, the products of the invention may be prepared by
procedures available to those in the art. A representative
synthesis procedure may be illustrated by the following Reaction
Schemes A-E: ##STR276##
As depicted in Reaction Scheme A, the aromatic esters (VI, VII, X
and XI) may be obtained by reaction of the appropriate acid
chloride (II, IX) derived from the substituted phenylalkanoic acids
I and VIII respectively and the desired phenol derivative (III, IV,
or V) in the presence of organic bases such as triethylamine,
pyridine or other commonly used reagents. Alternately, a solution
of the acid (I or VIII) and the phenol component (III or V) may be
treated with any of the carbodiimides (dicyclohexylcarbodiimide
[DCC] for example) already in use in the field of synthetic organic
chemistry to afford the corresponding aromatic esters (VI, X). In
the instances where the phenolic ester (V) is utilized above, the
benzyloxymethylene (BOM) protecting group is removed subsequent to
the coupling reaction to afford the free carboxylic acid
derivatives (VII, XI). The BOM groups which may be utilized to
prevent undesirable side reactions between the carboxylic acid
moiety of the phenol (IV) and the acid chlorides (II, IX) or the
nascent symmetrical anhydrides present during the coupling
reactions.
It will be evident to those skilled in the art that each of the
aforementioned reactions may require slightly different conditions,
dependent on the reactants involved, to obtain the best yields of
the desired products. In certain cases, for example, the
substituent R.sub.3 may be incompatible with some of the reagents
utilized in the overall reaction pathway. In those instances, an
appropriate protecting group must be chosen for R.sub.3 to prevent
undesired side reactions. For example, if R.sub.3 is hydroxy,
protection as the t-butyldimethylsilyl ether or benzyl ether will
allow the reaction sequence to proceed as specified. The conditions
for introducing and removing protecting groups, whether or not such
protecting groups are needed, are known to anyone skilled in the
art.
In cases where the phenol components bear a substituent containing
a sulfur atom directly attached to the aromatic ring (IV, V, III
with R.sub.4 =SCH.sub.3), the corresponding esters (VI, VII, X, XI)
may be oxidized to the respective sulfoxides (XII, XIV, XVI, XVIII)
by treatment with one equivalent of hydrogen peroxide or to the
sulfones (XIII, XV, XVII, XIX) by oxidation with excess peroxide as
described in Reaction Scheme B. The sulfones (XIII, XV, XVII, XIX)
are obtained directly from the sulfides (VI, VII, X, XI) without
isolation of the intermediate sulfoxides formed initially in the
presence of excess peroxide.
The phenolic compounds (III) are available commercially. The other
derivatives (IV, V) may be synthesized from readily available
starting materials as described in Reaction Scheme C.
4-Hydroxythiophenol (XX) may be oxidized to the disulfide (XXI) in
high yield. Subsequent masking of the hydroxyls of (XXI) with
suitable protecting groups (tert-butyldimethylsilyl, for example)
may be effected by treatment of the disulfide (XXI) with two
equivalents of tert-butyldimethylsilylchloride in the presence of
imidazole in DMF. There are numerous examples of protecting groups
for phenolic moieties published in the general synthetic chemistry
literature (see Greene, T. W., "Protective Groups in Organic
Synthesis", John Wiley and Sons, 1981). It is contemplated that
other available protecting groups could function similarly to the
tert-butyldimethylsilyl example cited above. These additional
protecting groups as well as the reaction conditions for
incorporating these groups at the appropriate point in the
synthesis are well known to practitioners skilled in the art.
Reaction of the protected disulfide (XXII) with
tri-n-butylphosphine in the presence of the appropriate alcohol
(XXIII or XXVI) provides the thioethers (XXIV and XXVII
respectively). Hydrolysis of the ester (XXIV) in aqueous KOH
results in cleavage of the silyl ether as well to give the phenolic
acid (IV). The BOM protected derivative (XXVII) may be selectively
desilylated with tetra-n-butylammonium fluoride in aqueous THF to
yield the BOM protected phenol (V). The commercially available
4-methylmercaptophenol (III, R.sub.4 =4-SCH.sub.3) may be converted
to the sulfoxide (III, R.sub.4 =4--S(O)CH.sub.3) and the sulfone
(III, R.sub.4 =4--S(O).sub.2 CH.sub.3) by oxidation with hydrogen
peroxide in acetic acid under the conditions specified in Reaction
Scheme C.
As illustrated in Reaction Scheme D, the appropriate phenylacetic
acids (XXVIII, XXXII) whether or not additionally substituted by
substituent R.sub.3 may be esterified by treatment with thionyl
chloride (SOCl.sub.2), or oxalyl chloride (C.sub.2 O.sub.2
Cl.sub.2) to generate the acid chloride which is subsequently
allowed to react with the appropriate alcohol (ROH) in the presence
of base or alternately by acid catalyzed esterification.
The phenylacetic acid esters (XXIX, XXXIII) thus obtained may be
alkylated at the .alpha.-position by generation of the enolate
anion with strong bases such as lithium diisopropylamide (LDA)
followed by reaction of the enolate with the appropriate alkyl
halide. The resulting 2-phenylalkanoates (XXX, XXXIV) may be
converted to the corresponding 2-phenylalkanoic acid derivatives
(I, VIII) by base hydrolysis of the alkyl esters and hydrogenolysis
of the benzyl esters.
The alkylated esters (XXX, XXXIV) may be alkylated further to yield
the .alpha.,.alpha.-dialkyl esters (XXXI, XXXV). Hydrolysis of the
esters (XXXI, XXXV) affords the dialkylated acids (I, VIII). If
R.sub.2 X=Br(CH.sub.2).sub.n Br then the corresponding esters (XXX,
XXXIV) have Br(CH.sub.2).sub.n -- as the R.sub.2 substituent and
subsequent treatment with LDA results in formation of the
1-phenylcycloalkane carboxylates (XXXI, XXXV, R.sub.1, R.sub.2
=--(CH.sub.2).sub.n --) which may be saponified to the
corresponding 1-phenylcycloalkane carboxylic acids (I, VIII,
R.sub.1, R.sub.2 =--(CH.sub.2).sub.n --).
A number of substituted phenylacetic acids and 2-phenylalkanoic
acids are commercially available and may be obtained directly for
use herein. Acids (I) and (VIII) bearing substituents R.sub.3 which
are not available may be synthesized by published procedures.
Reaction Scheme E describes some of the many examples of these
types of procedures which are known to those skilled in the
art.
Benzene derivatives (XXXVI, XXXIX) may be acylated by the Friedel
Crafts procedure to give arylketones. The substituted acetophenones
(XXXVII, XL) may be transformed to the phenylacetic acids (XXVIII,
XXXII) by the Wilgerodt-Kindler reaction sequence. The
butyrophenone derivatives (XXXVIII, XLI) may be oxidatively
rearranged to the phenylbutyric acids (I, VIII, [R.sub.2 =C.sub.2
H.sub.5 ]) by commonly used techniques. Additionally, available
phenylalkanoic acids (I) may be nitrated to provide the 4-nitro
derivative (I, R.sub.3 =4--NO.sub.2). Reduction of the nitro
substituent gives the amino compound (I, R.sub.3 =4--NH.sub.2)
which may be acylated to afford the amides (I, R.sub.3
=4--NHCOR.sub.8).
It will be evident to one skilled in this field of chemistry that
there are additional generally available methods of synthesizing
the compounds of the invention.
The following examples are given to illustrate the preparation of
specific compounds according to the invention:
EXAMPLE 1
Synthesis of 4-Nitrophenyl 2-(4'-Methoxyphenyl)butyrate (34)
Oxalyl chloride (12 mL of a 2.0M solution in CH.sub.2 Cl.sub.2) was
added under nitrogen to a solution of 2-(4'-methoxyphenyl)butyric
acid (4.66 g, 24 mmol) in 25 mL of CH.sub.2 Cl.sub.2 and stirred at
room temperature overnight. The volatiles were removed under vacuum
and the residue was distilled to afford 4.46 g (87%) of pure
2-(4'-methoxyphenyl)butyryl chloride. .sup.1 H NMR (CDCl.sub.3)
.delta.0.919 (t, 3H, J=7.4 Hz), 1.78-1.92 (m, 1H), 2.13-2.27 (m,
1H), 3.81 (s, 3H), 3.83 (t, 1H, J=7.6 Hz), 6.91 (d, 2H, J=8.8 Hz),
7.21 (d, 2H, J=8.7 Hz); .sup.13 C NMR (CDCl.sub.3) .delta.11.43,
26.26, 55.14, 64.21, 114.50, 127.86, 129.65, 159.75, 175.48.
The acid chloride (1.06 g, 5.0 mmol) was added to a mixture of
4-nitrophenol (0.696 g, 5.0 mmol) and pyridine (0.395 g, 5 mmol) in
5 mL of THF under N.sub.2 and stirred overnight at room
temperature. The solution was filtered and concentrated under
vacuum to give a yellow residue which was chromatographed on a
flash silica gel column (CH.sub.2 Cl.sub.2) to afford 1.46 g (92%)
of the desired nitrophenyl ester. .sup.1 H NMR (CDCl.sub.3)
.delta.0.988 (t, 3H, J=7.3 Hz), 1.83-1.97 (m, 1H), 2.13-2.28 (m,
1H), 3.67 (t, 1H, J=7.7 Hz), 3.82 (s, 3H), 6.92 (d, 2H, J=8.4 Hz),
7.18 (d, 2H, J=9.3 Hz), 7.31 (d, 2H, J=8.4 Hz), 8.23 (d, 2H, J=9.2
Hz).
EXAMPLE 2
Synthesis of 4-Methylmercaptophenyl 2-(4'-Methoxyphenyl)butyrate
(35)
To a stirred solution of 4-methylmercaptophenol (0.701 g, 5.0 mmol)
and pyridine (0.395 g, 5.0 mmol) in 5 mL of THF under N.sub.2 was
added a solution of 2-(4'-methoxyphenyl)butyryl chloride (1.06 g,
5.0 mmol) in 5 mL of THF. After stirring at room temperature
overnight, the precipitated pyridinium hydrochloride was filtered
off and the filtrate evaporated to give 1.76 g of crude ester.
Kugelrohr distillation afforded 1.53 g of the pure ester (95%
yield). .sup.1 H NMR (CDCl.sub.3) .delta.0.974 (t, 3H, J=7.4 Hz),
1.80-1.93 (m, 1H), 2.13-2.25 (m, 1H), 2.45 (s, 3H), 3.63 (t, 1H,
J=7.7 Hz), 3.81 (s, 3H), 6.90 (d, 2H, J=8.7 Hz), 6.92 (d, 2H, J=8.6
Hz), 7.23 (d, 2H, J= 8.7 Hz), 7.31 (d, 2H, J=8.7 Hz); .sup.13 C NMR
(CDCl.sub.3) .delta.11.84, 16.25, 26.49, 52.46, 55.13, 114.19,
122.06, 128.09, 129.17, 130.69, 135.68, 148.77, 159.15, 173.15.
EXAMPLE 3
Synthesis of 4-Methylsulfinylphenyl 2-(4'-Methoxyphenyl)butyrate
(36)
4-Methylmercaptophenyl 2-(4'-methoxyphenyl) butyrate (6.0 g, 19
mmol) in 63 g of glacial acetic acid was treated with 3.2 mL of 30%
H.sub.2 O.sub.2. The reaction was followed by TLC (silica, CH.sub.2
Cl.sub.2) until all of the starting material was consumed. The
product sulfoxide was extracted into ether. The ether layer was
washed with H.sub.2 O followed by saturated sodium bicarbonate and
then dried over anhydrous potassium carbonate for 16 hours. The
solution was filtered and evaporated under vacuum to give the pure
product (5.2 g, 82%). .sup.1 H NMR (CDCl.sub.3) .delta.0.981 (t,
3H), 1.80-1.95 (m, 1H), 2.12-2.28 (m, 1H), 2.69 (s, 3H), 3.66 (t,
1H), 3.80 (s, 3H), 6.91 (d, 2H), 7.16 (d, 2H), 7.31 (d, 2H), 7.63
(d, 2H); .sup.13 C NMR (CDCl.sub.3) .delta.11.78, 26.33, 43.91,
52.39, 55.10, 114.24, 122.75, 124.93, 129.12, 130.25, 142.88,
153.06, 159.22, 172.75.
EXAMPLE 4
Synthesis of 4-Methylsulfonylphenyl 2-(4'-Methoxyphenyl)butyrate
(37)
4-Methylmercaptophenyl 2-(4'-methoxyphenyl) butyrate (10.0 g, 31.6
mmol) was dissolved in 32 mL of glacial acetic acid, 30% H.sub.2
O.sub.2 (32 mL) was added and the solution stirred for 72 hours.
The reaction mixture was poured into 250 mL of ice water and
stirred for 30 minutes until all of the ice had melted. The white
solid was filtered off and washed with water until the filtrate was
neutral. The product was dried under vacuum to give 10.5 g (95%) of
the desired compound. .sup.1 H NMR (CDCl.sub.3) .delta.0.986 (t,
3H, J=7.5 Hz), 1.83-1.96 (m, 1H), 2.13-2.27 (m, 1H), 3.04 (s, 3H),
3.67 (t, 1H, J=7.7 Hz), 3.82 (s, 3H), 6.92 (d, 2H, J=8.7 Hz), 7.21
(d, 2H, J=8.7 Hz), 7.31 (d, 2H, J=8.7 Hz), 7.94 (d, 2H, J=8.7 Hz);
.sup.13 C NMR (CDCl.sub.3) .delta.12.10, 26.61, 44.78, 52.76,
55.47, 114.66, 123.05, 129.47, 129.58, 130.35, 138.17, 155.45,
159.65, 172.79.
EXAMPLE 5
Synthesis of 2,2-Dimethyl-3-(4'-Hydroxyphenylthio)propionic acid
(IV)
A) A solution of bromine (95 g, 0.59 mol) in 500 mL of CH.sub.2
Cl.sub.2 was added dropwise to a solution of 4-hydroxythiophenol
(150 g, 1.19 mol) in 500 mL of CH.sub.2 Cl.sub.2 until the orange
color persisted. The reaction mixture was stirred overnight, 1 L of
petroleum ether was added and the solid was filtered and dried
under vacuum to give 98.5 g (67%) of 4'-hydroxyphenyldisulfide.
B) 4'-Tert-butyldimethylsilyloxyphenyldisulfide (XXII).
Solid t-butyldimethylsilylchloride (132.6 g, 0.88 mol) was added to
a stirred solution of 4'-hydroxyphenyldisulfide (100.1 g, 0.40 mol)
and imidazole (119.8 g, 1.76 mol) in 500 mL of DMF under nitrogen.
After 2 hours the reaction mixture was poured into 750 mL of
H.sub.2 O and extracted with ether (3.times.300 mL). The combined
ether layers were washed with H.sub.2 O, dried over MgSO.sub.4 and
evaporated to give 201.0 g of the product as a yellow liquid. The
crude product can be purified further by vacuum distillation or
chromatography on silica gel (pet. ether) to give the desired
compound in a near quantitative yield.
C) Methyl
2,2-dimethyl-3-(4'-tert-butyldimethylsilyloxyphenylthio)propionate
(XXIV).
Methyl 2,2-dimethyl-3-hydroxypropionate (4.23 g, 32 mmol),
4'-tert-butyldimethylsilyloxyphenyldisulfide (14.36 g, 30 mmol) and
tri-n-butylphosphine (6.06 g, 30 mmol) were heated together under
reflux for 48 hours under a nitrogen atmosphere. The reaction
mixture was concentrated under vacuum, H.sub.2 O was added and the
mixture extracted with pet. ether. After drying over MgSO.sub.4,
the solution was concentrated to give 20.72 g of a clear liquid.
The product was isolated by chromatography on silica gel. The
by-product 4-tert-butyldimethylsilyloxythiophenol (7.35 g, 102%)
eluted first with pet. ether. The thioether product (7.98 g, 75%)
was eluted with 50:50 pet. ether/CH.sub.2 Cl.sub.2. .sup.1 H NMR
(CDCl.sub.3) .delta. 0.175 (s, 6H), 0.965 (s, 9H), 1.25 (s, 6H),
3.08 (s, 2H), 3.55 (s, 3H), 6.75 (d, 2H, J=8.4 Hz), 7.29 (d, 2 H,
J=8.4 Hz); .sup.13 C NMR (CDCl.sub.3) .delta.-4.81, 17.91, 24.55,
25.39, 43.81, 46.52, 51.62, 120.72, 128.21, 133.36, 155.18,
176.98.
D) 2,2-Dimethyl-3-(4'-hydroxyphenylthio)-propionic acid (IV).
Methyl
2,2-dimethyl-3-(4'-tert-butyldimethylsilyloxyphenylthio)propionate
(7.09 g, 20 mmol) was added to KOH (6.73 g, 120 mmol) in 40 mL of
H.sub.2 O and the mixture was heated to reflux overnight. The
reaction mixture was cooled to room temperature, diluted with 40 mL
of H.sub.2 O and extracted with ether. The aqueous layer was
separated, acidified to pH=2 and extracted with ether (3.times.100
mL). The combined ether layers were dried over anhydrous
MgSO.sub.4, filtered and evaporated to give 3.72 g (82%) of
2,2-dimethyl-3-(4'-hydroxyphenylthio)propionic acid as a white
solid. .sup.1 H NMR (CD.sub.3 COCD.sub.3) .delta.1.25 (s, 6H), 3.11
(s, 2H), 6.80 (d, 2H, J=8.4 Hz), 7.31 (d, 2H, J=8.7 Hz), 8.6 (br s,
1H, --OH); .sup.13 C NMR (CD.sub.3 COCD.sub.3) .delta.25.13, 44.53,
47.63, 117.21, 127.46, 134.67, 158.18, 178.41.
EXAMPLE 6
Synthesis of 4-(2'-Carboxy-2'-methylpropylmercapto)phenyl
2-Phenylbutyrate (323)
A) 2-Phenylbutyryl chloride
Thionyl chloride (0.12 mol) in 60 mL of CH.sub.2 Cl.sub.2 was added
to a stirred solution of 2-phenylbutyric acid (16.4 g, 0.10 mol). A
catalytic amount of DMF was added and the reaction was allowed to
continue overnight at room temperature. The volatiles were removed
under vacuum and the residual liquid was vacuum distilled to yield
12.4 g (68%) of 2-phenylbutyryl chloride. IR (neat) 1798 cm.sup.-1
(C.dbd.O).
B) 4-(2'-Carboxy-2'-methylpropylmercapto)-phenyl
2-phenylbutyrate.
A solution of 2-phenylbutyryl chloride (1.19 g, 6.5 mmol) in 5 mL
of THF was added to a stirred solution of
2,2-dimethyl-3-(4'-hydroxyphenylthio)-propionic acid (1.36 g, 6.0
mmol) and pyridine (1.03 g, 13.0 mmol) in 10 mL of THF under
N.sub.2. After 6 days, 30 mL of ether was added and the reaction
mixture filtered into a separatory funnel. The organic layer was
washed with 0.5N HCl (2.times.15 mL), saturated NaCl (15 mL), 1:9
saturated NaHCO.sub.3 /H.sub.2 O (2.times.15 mL), saturated NaCl
(15 mL) and dried over anhydrous MgSO.sub.4. Filtration and
evaporation provided 1.96 g (88%) of the desired ester. .sup.1 H
NMR (CDCl.sub.3) .delta.0.974 (t, 3H, J=7.2 Hz), 1.27 (s, 6H),
1.82-1.96 (m, 1H), 2.13-2.28 (m, 1H), 3.13 (s, 2H), 3.67 (t, 1H,
J=7.5 Hz), 6.90 (d, 2H, J=8.7 Hz), 7.30-7.38 (ArH, 7H), 11.9 (br s,
--OH); .sup.13 C NMR (CDCl.sub.3) .delta.11.84, 24.30, 26.45,
43.73, 44.95, 53.27, 122.06, 127.58, 128.01, 128.88, 131.66,
134.27, 138.58, 149.65, 172.75, 183.22.
EXAMPLE 7
Synthesis of 4-(2'-Carboxy-2'-methylpropylsulfinyl)phenyl
2-Phenylbutyrate (324)
To a solution of 4-(2'-carboxy-2'-methylpropylmercapto)phenyl
2-phenylbutyrate (745 mg, 2 mmol) in 1 mL of glacial acetic acid
was added 0.25 mL of 30% H.sub.2 O.sub.2. Additional 0.25 mL
aliquots of 30% H.sub.2 O.sub.2 were added at one-half hour
intervals until TLC indicated complete consumption of the starting
material. The reaction was quenched with 20 mL of H.sub.2 O,
extracted with Et.sub.2 O (2.times.25 mL), dried over anhydrous
MgSO.sub.4 and evaporated to give the sulfoxide containing a
residual amount of acetic acid. The residue was suspended in 20 mL
of H.sub.2 O, the mixture was shell frozen and lyophilized to give
506 mg (65%) of the pure product sulfoxide. .sup.1 H NMR
(CDCl.sub.3) .delta.1.00 (t, 3H, J=7.4 Hz), 1.42 (s, 3H), 1.54 (s,
3H), 1.85-1.99 (m, 1H), 2.16-2.31 (m, 1H), 3.07 (s, 2H), 3.72 (t,
1H, J=7.6 Hz), 7.17 (d, 2H, J=8.7 Hz), 7.32-7.40 (ArH, 5H), 7.70
(d, 2H, J=8.7 Hz), 11.17 (br s, --OH); .sup.13 C NMR (CDCl.sub.3)
.delta. 11.83, 24.54, 25.71, 26.39, 41.73, 53.26, 68.81, 122.75,
125.60, 127.73, 128.10, 128.98, 138.30, 141.34, 153.11, 172.49,
180.33.
EXAMPLE 8
Synthesis of 4-(2'-Carboxy-2'-methylpropylsulfonyl)phenyl
2-Phenylbutyrate (325)
To a stirred solution of
4-(2'-carboxy-2'-methylpropylmercapto)phenyl 2-phenylbutyrate (745
mg, 2 mmol) in 4 mL of glacial acetic acid was added 4 mL of 30%
hydrogen peroxide. After 36 hours the reaction was quenched with 20
mL of H.sub.2 O, extracted with Et.sub.2 O (2.times.25 mL), dried
over anhydrous MgSO.sub.4 and evaporated under vacuum. The residue
was suspended in H.sub.2 O, shell frozen and lyophilized to afford
728 mg (90%) of pure 4-(2'-carboxy-2'-methylpropylsulfonyl)phenyl
2-phenylbutyrate. .sup.1 H NMR (CDCl.sub.3) .delta.0.992 (t, 3H,
J=7.4 Hz), 1.46 (s, 6H), 1.85-2.00 (m, 1H), 2.16-2.30 (m, 1H), 3.47
(s, 2H), 3.72 (t, 1H, J=7.6 Hz), 7.20 (d, 2H, J=8.7 Hz), 7.32-7.38
(ArH, 5H), 7.92 (d, 2H, J=8.7 Hz), 10.9 (br s, --OH); .sup.13 C NMR
(CDCl.sub.3) .delta.11.79, 24.94, 26.33, 41.21, 53.26, 64.32,
122.63, 127.83, 128.08, 129.03, 129.64, 138.07, 138.33, 155.06,
172.18, 181.47.
EXAMPLE 9
Synthesis of Benzyloxymethyl
2,2-Dimethyl-3-(4'-Hydroxyphenylthio)propionate (V)
A) Benzyloxymethyl 2,2-dimethyl-3-hydroxypropionate (XXVI).
A solution of methyl 2,2-dimethyl-3-hydroxypropionate (25.0 g,
0.189 mol) in 100 mL of MeOH was treated with a solution of KOH
(11.7 g, 0.208 mol) in 50 mL of H.sub.2 O and the resulting mixture
stirred at room temperature for 5 hours. The reaction mixture was
heated under reflux for 30 minutes, methanol was distilled and the
remaining solution shell frozen and lyophilized to give 26.7 g
(90.6%) of potassium 2,2-dimethyl-3-hydroxypropionate as a white
solid. The potassium salt (13.0 g, 0.083 mol) was suspended in 100
mL of dry DMF and chloromethylenebenzylether (14.3 g, 0.092 mol)
was added. After stirring 48 hours at room temperature the mixture
was quenched with 100 mL of H.sub.2 O and extracted with Et.sub.2 O
(200 mL). The ether layer was separated, washed with H.sub.2 O
(3.times.100 mL), saturated NaCl (100 mL) and dried over
MgSO.sub.4. Evaporation and distillation of the residue gave 13.6 g
(73%) of benzyloxymethyl 2,2-dimethyl-3-hydroxypropionate as a
clear liquid.
B) Benzyloxymethyl
2,2-dimethyl-3-(4'-tert-butyldimethylsilyloxyphenylthio)propionate
(XXVII).
Benzyloxymethyl 2,2-dimethyl-3-hydroxypropionate (2.40 g, 10.2
mmol), 4'-tert-butyldimethylsilyloxyphenyldisulfide (4.89 g, 10.2
mmol) and tri-n-butylphosphine (2.07 g, 10.2 mmol) were heated
together under reflux in 30 mL of THF under a nitrogen atmosphere
for 18 hours. The reaction mixture was diluted with ether, washed
with H.sub.2 O (3.times.100 mL), dried over anhydrous MgSO.sub.4
and evaporated. The residue was chromatographed on silica gel (pet.
ether/CH.sub.2 Cl.sub.2) to give 1.82 g (39%) of the desired
product. .sup.1 H NMR (CDCl.sub.3) .delta.0.181 (s, 6H), 0.975 (s,
9H), 1.28 (s, 6H), 3.12 (s, 2H), 4.67 (s, 2H), 5.28 (s, 2H), 6.75
(d, 2H, J=8.7 Hz), 7.2-7.4 (m, 7H); .sup.13 C NMR (CDCl.sub.3)
.delta.-4.80, 17.91, 24.45, 25.39, 44.15, 46.25, 71.70, 88.63,
120.79, 127.87, 128.03, 128.09, 128.18, 128.59, 133.27, 137.16,
155.21, 176.07.
C) Benzyloxymethyl 2,2-dimethyl-3-(4'-hydroxyphenylthio)propionate
(V).
A 1.0M solution of tetra-n-butylammonium fluoride in THF (3.6 mL,
1.1 equiv) was added to the tert-butyldimethylsilylether (XXVII)
(1.50 g, 3.3 mmol) in 25 mL of THF at -10.degree. C. After 1 hour
the reaction was acidified with saturated ammonium chloride (25 mL)
and extracted with ether. The ether layer was washed with H.sub.2
O, saturated NaCl, dried over anhydrous MgSO.sub.4 and
concentrated. The residue was chromatographed on silica gel to give
0.92 g (82%) of V. .sup.1 H NMR (CDCl.sub.3) .delta.1.27 (s, 6H),
3.10 (s, 2H), 4.67 (s, 2H), 5.10 (br s, 1H), 5.28 (s, 2H), 6.74 (d,
2H, J=8.4 Hz), 7.2-7.4 (m, 7H); .sup.13 C NMR (CDCl.sub.3)
.delta.25.10, 44.82, 47.19, 72.41, 89.38, 116.72, 128.07, 128.68,
128.76, 129.24, 134.49, 137.74, 155.91, 176.91.
EXAMPLE 10
Synthesis of 4-(2'-Carboxy-2'-methylpropylmercapto)phenyl
2-(4'-Methoxyphenyl)isobutyrate (308)
A solution of 2-(4'-methoxyphenyl)isobutyric acid (1.5 g, 7.7 mmol)
in CH.sub.2 Cl.sub.2 was treated with a 2.0M oxalyl chloride
solution in CH.sub.2 Cl.sub.2 (7.7 mL, 15.4 mmol) and a drop of
DMF. After stirring overnight at room temperature, the volatiles
were removed under vacuum and the residue dissolved in dry THF. The
resulting solution of 2-(4'-methoxyphenyl)isobutyryl chloride was
added to a stirred solution of V (2.68 g, 7.7 mmol) and pyridine
(0.73 g, 9.3 mmol) in THF and stirred overnight. The reaction
mixture was concentrated under vacuum, the residue dissolved in
ether and washed with H.sub.2 O. The organic layer was washed
subsequently with dilute HCl, dilute bicarbonate, H.sub.2 O and
dried over anhydrous MgSO.sub.4. The product was isolated by
preparative HPLC to afford 1.1 g (27%) of the benzyloxymethyl
protected ester. The benzyloxymethyl group was removed by treatment
with 40 mL of 6N HCl/40 mL of THF for 1 hour. Saturated NaCl was
added, the reaction mixture extracted with ether, washed with
dilute bicarbonate solution, dried over anhydrous MgSO.sub.4 and
evaporated to give 0.75 g (89%) of
4-(2'-carboxy-2'-methylpropylmercapto)phenyl
2-(4'-methoxyphenyl)isobutyrate. .sup.1 H NMR (CDCl.sub.3)
.delta.1.27 (s, 6H), 1.68 (s, 6H), 3.14 (s, 2H), 3.81 (s, 3H),
6.86-6.93 (m, 4H, ArH), 7.35-7.38 (m, 4H, ArH); .sup.13 C NMR
(CDCl.sub.3) .delta.24.36, 26.22, 43.75, 45.08, 45.91, 55.16,
114.00, 122.04, 126.93, 131.85, 134.10, 136.18, 150.02, 158.69,
175.79, 182.77.
EXAMPLE 11
Synthesis of 4-(2'-Carboxy-2'-methylpropylmercapto)phenyl
2-(1',2',3',4'-Tetrahydro-6'-naphthyl)butyrate (389)
A solution of 2-(1',2',3',4'-tetrahydro-6'-naphthyl)butyryl
chloride (4.0 mmol) in 16 mL of dry THF was added to a solution of
IV (814 mg, 3.6 mmol) and pyridine (790 mg, 10 mmol) in 20 mL of
dry THF and stirred under N.sub.2 for 3 days. The THF was removed
at the rotary evaporator and the residue dissolved in ether. The
ether layer was washed successively with H.sub.2 O (100 mL), dilute
HCl, dilute NaHCO.sub.3, dried over MgSO.sub.4 and concentrated to
give 1.35 g (79%) of the desired ester. .sup.1 H NMR (CDCl.sub.3)
.delta.0.955 (t, 3H), 1.28 (s, 6H), 1.75-1.98 (m, s, 4H), 2.10-2.31
(m, 1H), 2.78 (br s, 4H), 3.14 (s, 2H), 3.59 (t, 1H), 6.92 (d, 2H),
7.01-7.19 (m, 3H, ArH), 7.37 (d, 2H), (--OH not observed); .sup.13
C NMR (CDCl.sub.3) .delta.11.96, 22.91, 23.95, 24.32, 26.59, 28.85,
29.21, 43.74, 45.04, 52.98, 122.16, 125.06, 128.75, 129.63, 131.74,
134.13, 135.63, 136.51, 137.66, 149.81, 173.00, 182.98.
EXAMPLE 12
Synthesis of 4-(Methylmercapto)phenyl 2-Phenylbutyrate (5)
To a flask containing 2-phenylbutyric acid (4.93 g, 30 mmol) in 40
mL of CH.sub.2 Cl.sub.2 at 0.degree. C. was added
dicyclohexylcarbodiimide (6.19 g, 30 mmol) in 30 mL of CH.sub.2
Cl.sub.2. Solid 4-methylmercaptophenol (4.21 g, 30 mmol) was added
and the suspension stirred at room temperature overnight. The
precipitated urea was filtered, the filtrate evaporated and the
residue chromatographed on silica gel (CH.sub.2 Cl.sub.2)to give
4-(methylmercapto)phenyl 2-phenylbutyrate (6.23 g, 73%) as a light
yellow oil which was crystallized from EtOH to give white crystals
(mp 28.0.degree.-28.5.degree. C.). .sup.1 H NMR (CDCl.sub.3)
.delta.0.98 (t, 3H, J=7.3 Hz), 1.89 (m, 1H), 2.22 (m, 1H), 3.68 (t,
1H, J=7.6 Hz), 6.92 (d, 2H, J=8.6 Hz), 7.22 (d, 2H, J= 8.6 Hz),
7.30-7.45 (m, 5H, ArH).
EXAMPLE 13
Synthesis of 4-(2'-Carboxy-2'-methylpropylmercapto)phenyl
2-(4'-Benzamidophenyl)butyrate (338)
A) 2-(4'-Nitrophenyl)butyric acid
A mixture of concentrated nitric acid (32 mL) and concentrated
sulfuric acid (32 mL) were cooled in an ice salt bath. Solid
2-phenylbutyric acid (16.42 g, 100 mmol) was added in small
portions maintaining the solution temperature below 10.degree. C.
The reaction was warmed to room temperature and allowed to stir for
1 hour. The product was isolated by pouring the reaction mixture
onto 150 mL of crushed ice, filtering the white solid and
recrystallizing from EtOH to give 14.5 g (69%) of the product as
white crystals.
B) 2-(4'-Aminophenyl)butyric acid
A solution of 2-(4'-nitrophenyl)butyric acid (6.99 g, 33.4 mmol) in
250 mL of EtOH and 0.5 g of 10% Pd-C was hydrogenated overnight at
55 psi. The solution was filtered and evaporated under vacuum to
give 5.50 g (92%) of the desired product.
C) 2-(4'-Benzamidophenyl)butyric acid
Benzoylchloride (7.84 g, 0.057 mol) was added dropwise to a
solution of 2-(4'-aminophenyl)butyric acid (10.0 g, 0.057 mol) and
pyridine (4.85 g, 0.061 mol) in 100 mL of THF at 0.degree. C. After
30 minutes the ice bath was removed and the reaction warmed to room
temperature. After 1 hour the suspension was diluted with 300 mL of
ether, washed with 10% HCl (3.times.50 mL), saturated NaCl (50 mL),
dried over MgSO.sub.4 and evaporated to give a brown solid.
Trituration with ether afforded 7.54 g (47.7%) of the product as a
white solid.
D) 4-(2'-Carboxy-2'-methylpropylmercapto)phenyl
2-(4'-benzamidophenyl)butyrate
Dicyclohexylcarbodiimide (1.44 g, 7.0 mmol) was added to a solution
of 2-(4'-benzamidophenyl)butyric acid (1.70 g, 6.0 mmol) and V
(2.08 g, 6.0 mmol) in 60 mL of CH.sub.2 Cl.sub.2 with stirring at
room temperature. After 3 days, 4-dimethylaminopyridine (0.10 g)
was added and the reaction was allowed to proceed an additional 24
hours. The reaction was quenched with 2 mL of acetic acid,
filtered, washed with H.sub.2 O (3.times.50 mL), saturated NaCl and
dried over anhydrous MgSO.sub.4. Removal of the solvent afforded
3.02 g (82%) of the product as a clear oil. The benzyloxymethyl
group was removed by treating the oil (1.60 g, 2.6 mmol) with 50 mL
of 6N HCl and 100 mL of THF at 0.degree. C. for 1 hour followed by
an additional 50 mL of THF and 50 mL of 6N HCL. After 1 hour the
reaction was quenched with 50 mL of saturated NaCl and extracted
with ether (300 mL). The ether layer was dried over MgSO.sub.4,
evaporated and the residue chromatographed to give the product as
an oil which crystallized from EtOAc/hexane as a white solid (0.66
g, 52%). .sup.1 H NMR (CDCl.sub.3) .delta.0.983 (t, 3H, J=7.5 Hz),
1.29 (s, 6H), 1.8-2.3 (m, 2H), 3.14 (s, 2H), 3.67 (t, 1H, J=7.8
Hz), 6.91 (d, 2H, J=8.7 Hz), 7.37 (d, 2H, J=8.1 Hz), 7.45-7.6 (m,
5H, ArH), 7.64 (d, 2H, J=8.1 Hz), 7.87 (d, 2H, J=7.2 Hz), 7.97 (s,
1H), (--OH not observed); .sup.13 C NMR (CDCl.sub.3) .delta.11.87,
24.41, 26.44, 43.75, 45.10, 52.78, 120.66, 122.12, 127.22, 128.89,
129.01, 131.89, 132.15, 134.28, 134.72, 135.04, 137.45, 149.64,
166.17, 172.90, 182.17.
As noted earlier, the present compounds demonstrate HLE inhibiting
activity which indicates that these compounds would be useful in
the treatment of such diseases as emphysema, arthritis,
atheriosclerosis or the like. For such uses, the compounds would be
administered by the usual routes, e.g. orally, intravenously,
subcutaneously, intraperitoneally or intramuscularly. For
emphysema, the compounds would be administered in therapeutically
effective amounts, usually orally or rectally, or as a mist for
bronchial inhalation.
The amount of compound used to inhibit HLE will vary with the
nature and extent of the condition involved. It is contemplated,
for example, that mists containing from 0.05 to 20% of the active
compound with dosages in the order of 2-100 mg per dosage unit
several times a day would provide a therapeutically effective
amount for the treatment of emphysema. Variations and adjustments
in the size and frequency of administration can be determined to
provide the desired HLE inhibition.
Pharmaceutical compositions containing the active compounds of the
invention may comprise tablets, capsules, solutions or suspensions
with conventional non-toxic pharmaceutically acceptable carriers.
These compositions may include the usual types of additives, e.g.
disintegrating or suspending agents or the like. Compounds selected
for intravenous use should be soluble in aqueous solutions, while
those used in, for example, oral formulations need not be
water-soluble.
Topical formulations are also contemplated for use in the treatment
of, for example, dermatitis and acne.
The compounds of the invention are extremely potent and highly
selective inhibitors of neutrophil elastase. The compounds also
appear to show adequate serum stability. The water solubility of
the compounds varies and it will be appreciated that the ultimate
mode of administration for each compound will depend, at least to
some extent, on the solubility of the compound involved.
In this regard, it appears that water solubility of the present
compounds may be improved, without undesirably affecting activity,
selectivity or serum stability, by appropriate selection of the
R.sub.4 substituent(s) on the phenyl ring of the Formula (VI)
compounds. These compounds may be viewed as made up of two
components, i.e. an acylating group and a leaving group introduced
by the acid and phenol reactants, respectively. The introduction of
particular solubilizing substituents R.sub.4 on the leaving group
to improve solubility in aqueous solutions or buffers without
undesirably affecting the activity of the compound is illustrated
by the following data which compares a representative series of
compounds with and without the modified leaving groups (TABLE
III).
TABLE III ______________________________________ ##STR277## PBS
I.sub.50 Solubility R.sub.4 .mu.m (mg/mL)
______________________________________ SCH.sub.3 9.000 0.010
S(O)CH.sub.3 0.600 0.600 S(O).sub.2 CH.sub.3 0.100 0.010 SCH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 0.892 .gtoreq.2.00 S(O)CH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 0.357 .gtoreq.2.00 S(O.sub.2)CH.sub.2
C(CH.sub.3).sub.2 CO.sub.2 H 0.141 .gtoreq.2.00
______________________________________
Without intending to be limited to any theory of operation or
function, it appears that the compounds of the invention bind to
the active site of neutrophil elastase. More particularly, it
appears that the acyl group binds to the S substrate position, i.e.
the valine or proline-valine region of the binding pocket and the
leaving group extends into the S' positions.
Representative compounds according to the invention have been
compared with a compound (Compound A) typifying the compounds
described in U.S. Pat. No. 4,801,610. The comparisons were directed
towards potency (represented by the I.sub.50 's for human
neutrophil elastase (HNE), porcine pancreatic elastase (PPE) and
alpha-chymotrypsin (.alpha.-CH)), specificity (represented by the
ratios of the I.sub.50 's (PPE/HNE and .alpha.-CH/HNE)) and the
ability to inhibit the digestion of extracellular matrix by
activated intact human neutrophils (expressed as a fraction of
control) of the compounds listed. The following results were
obtained (TABLE IV):
TABLE IV
__________________________________________________________________________
ECM (Frac- tion of Con- .alpha.- HNE trol 10 .mu.M PPE (I.sub.50)
Chymotrypsin (I.sub.50) Compound Structure I.sub.50 Inhibitor) HNE
(I.sub.50) HNE(I.sub.50)
__________________________________________________________________________
Compound A ##STR278## 0.129 0.86 18.84 146.10 3 ##STR279## 0.025
0.57 129.6 176.0 37 ##STR280## 0.031 0.62 38.6 106.2 188 ##STR281##
0.028 0.40 21.9 732.8 289 ##STR282## 0.090 0.43 1294.0 1624.6 346
##STR283## 0.039 0.28 8.71 45.9 384 ##STR284## 0.055 0.62 29.1
131.8
__________________________________________________________________________
##STR285##
The above data indicate that the introduction of an aromatic ring
substituent on the alpha carbon according to the invention will
improve potency relative to a compound bearing a simple pivaloyl
group (Compounds 3, 37, 188, 289, 346 and 384 versus Compound A).
In addition, an aromatic substituent in place of a methyl group on
the alpha carbon also significantly improves relative specificity,
particularly with regard to porcine pancreatic elastase (PPE)
(Compound 289 versus Compound A). Similarly, in the extracellular
matrix (ECM) assay, which compares the ability of a compound to
inhibit an intact neutrophil's digestion of extacellular matrix
proteins, all of the herein disclosed compounds were more effective
than the reference Compound A.
The following tests have been used to determine the activity of the
compounds of the present invention:
Potency (I.sub.50 Determination)
Reagents:
A) 0.075M sodium phosphate, 20% dimethyl sulfoxide (DMSO), pH
7.7=substrate and inhibitor buffer
B) 0.075M sodium phosphate no DMSO, pH 7.7=inhibitor buffer
C) 10 mM human neutrophil elastase (HNE) substrate
=N-methoxysuccinyl-ala-ala-pro-val-pNA in DMSO
D) 0.01M sodium acetate, 20% DMSO, pH 5.5=enzyme buffer
(dilution)
E) 0.01M sodium acetate, pH 5.5=enzyme buffer (storage)
F) HNE (1 mg) dissolved in 1 mL of reagent E for storage at
-20.degree. C.
Make a 10 mM stock of the inhibitor in DMSO. Dilute an aliquot (10
.mu.L) up to 1.0 mL in reagent A (100 .mu.M). Serially dilute 100
.mu.L of the 100 .mu.M stock to 10.0, 1.0, 0.1, 0.01 .mu.M in
reagent A. Apply 100 .mu.L of the diluted material to the wells of
a 96-well plate. Dilute an aliquot of reagent F 1:150 in reagent D,
apply 50 .mu.L aliquots to the indicated wells and incubate for 7
minutes at room temperature.
The HNE substrate solution is made by taking 100 .mu.L of reagent C
into 500 .mu.L of reagent A and 400 .mu.L of reagent B. After the 7
minutes of incubation, the substrate (50 .mu.L) is applied to each
well. The HNE catalyzed reaction is then monitored
spectrophotometrically at 405 nm using an ELISA plate reader
machine (UVMAX, Molecular Devices) which processes the raw data
with an on-board kinetics program. The enzyme activity is plotted
against different inhibitor concentrations and the I.sub.50 value
is determined by using a curve fitting software program. Once the
"screening" I.sub.50 has been approximated, a more precise I.sub.50
value can be obtained by examination of inhibitor concentrations
around this value.
Specificity Determination
Reagents:
1) Porcine Pancreatic Elastase (PPE) 1 mg/mL in 0.01M sodium
acetate, pH 5.5. An aliquot of this stock solution is diluted 1:20
in 0.01M sodium acetate, 20% DMSO, 10 mM CaCl.sub.2, pH 5.5.
2) .alpha.-Chymotrypsin (.alpha.-CH) 1 mg/mL in 0.01M sodium
acetate, pH 5.5. An aliquot of this stock solution is diluted 1:85
in 0.01M sodium acetate, 20% DMSO, 10 mM CaCl.sub.2, pH 5.5, 0.005%
triton X-100 detergent.
3) PPE substrate: N-succinyl-ala-ala-ala-pNA 20 mM stock in
DMSO.
4) .alpha.-CH substrate: N-succinyl-ala-ala-pro-leu-pNA 20 mM stock
in DMSO.
Inhibitor, substrate buffer: 0.1M tris-HCl, 0.01M CaCl.sub.2,
0.005% triton X-100, 20% DMSO, pH 7.7.
Production of Extracellular Matrix (ECM)
1. Rat smooth muscle cells (R22), grown in a stock culture are
detached from the flask surface with trypsin/EDTA solution, washed
with fetal calf serum-containing MEM and seeded at a concentration
of 50,000 cells per well (1 mL/well) using a 24-well tissue culture
plate.
2. Culture medium: Eagle's MEM with Earle's salts
1% penicillin/streptomycin
1% glutamine
10% heat inactivated fetal calf serum
2% tryptose phosphate broth
3. The cells are then grown to confluence (3-4 days), the medium
removed and new medium containing .sup.3 H-proline (500 .mu.Ci/L)
added.
4. At the same time the radioactive medium is added, 1
drop/well/day of an ascorbic acid solution (1.28 mg/mL of Hank's
balanced salt solution) is added.
5. Fresh culture medium containing .sup.3 H-proline is added after
5 days and the culture continued for a total of 8-10 days.
6. The medium is then removed and the wells washed twice with
phosphate buffered saline (PBS). The cells are lysed with 1 mL of
25 mM NH.sub.4 OH for approximately 3-5 minutes, the solution is
removed and the wells allowed to air dry (uncovered under UV light)
overnight.
7. The wells are rinsed 3 times with PBS and frozen with 1 mL of
PBS per well at -20.degree. C.
8. When plates are required for the assay, they are thawed for 2 h
at 37.degree. C. and rinsed once with Hank's balanced salt
solution.
Human Neutrophil Isolation and ECM Digestion Assay
1. Blood is drawn into heparinized syringes (1 mL/25 mL of
blood).
2. Heparinized blood (25 mL) is then added to 15 mL of Hetastarch,
gently mixed and the red cells allowed to settle for 25-30 minutes
at room temperature.
3. The red cell free supernatant is then layered on top of a
discontinous Percoll gradient (3 mL 74% Percoll; 3 mL 55%).
4. The tubes are then centrifuged at 1500.times.g for 20 minutes in
a non-refrigerated centrifuge.
5. PMNs are then collected from the 74/55% interface, diluted and
washed 2 times with saline.
6. If red cells are present, they are then lysed with deionized
water for 15 seconds. Salt solution is added to return the salt
concentration to 0.9% saline and the PMNs collected by
centrifugation.
7. PMNs are then resuspended in Dulbecco's MEM containing 1%
glutamine and 1% penicillin/streptomycin and counted using crystal
violet dye.
8. The PMN concentration is then adjusted to 10.sup.6 cells/mL and
aliquoted into the wells (1 mL/well) of the previously described
.sup.3 H-proline ECM culture plate.
9. The cells are allowed to settle for 15 minutes and the inhibitor
added, followed immediately by phorbol myristate acetate (PMA)
(final concentration=10 nM).
10. The plates are then incubated at 37.degree. C. and 100 .mu.L
aliquots of supernatant removed at various time points. The
solubilized radioactivity is measured by liquid scintillation
counting.
11. Radioactivity (counts/minute) from the background (no PMN)
wells are then subtracted from the measured counts.
12. Inhibition is assessed by determining the ratio of counts found
in the experimental wells (inhibitor added) to the counts obtained
from the wells in which no inhibitor was added: ##EQU1##
It will be appreciated that various modifications may be made in
the invention described herein without departing from the spirit
and scope of the invention as defined in the following claims
wherein:
* * * * *